WO2024222929A1

WO2024222929A1 - Gene therapy compositions and methods for reprogramming glial cells

Info

Publication number: WO2024222929A1
Application number: PCT/CN2024/090245
Authority: WO
Inventors: Jie Xu; Yuchen Chen; Meng Liu; Russell ADDIS; Pratima PANDEY
Original assignee: Neuexcell Therapeutics Suzhou Co Ltd
Current assignee: Neuexcell Therapeutics Suzhou Co Ltd
Priority date: 2023-04-28
Filing date: 2024-04-27
Publication date: 2024-10-31
Anticipated expiration: 2025-10-28

Abstract

Methods and compositions for producing new neurons in the brain in vivo are provided according to aspects of the present invention which include introducing NeuroD1 alone or in combination with one or more protein factors into a glial cell, particularly into a reactive astrocyte or NG2 cell, thereby converting the reactive glial cell to a functional neuron. Related methods of producing a neuronal phenotype in a glial cell are also provided according to aspects of the present invention which include expressing NeuroDl alone or in combination with one or more protein factors in the glial cell, wherein expressing NeuroDl includes delivering one or more nucleic acid molecules, particularly RNA molecules, formulated as part of lipid nanoparticles to the glial cell.

Description

GENE THERAPY COMPOSITIONS AND METHODS FOR REPROGRAMMING GLIAL CELLS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority to International Patent Application No.: PCT/CN2023/091637 filed on April 28, 2023, the contents of each of which is incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a computer readable Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14770-041-228_SEQLISTING. xml” , was created on April 25, 2024, and is 144, 200 bytes in size.

1. FIELD

The present disclosure generally relates to nucleic acid molecules encoding protein factors that can be used for reprogramming glial cells to functional neuronal cells, and compositions and methods for delivering the nucleic acid molecules to the glial cells both in vitro and in vivo.

2. BACKGROUND

The central nervous system in mammals is largely unable to regenerate itself following injury. Neurons are often killed or injured as a result of an injury or neurological condition, such as a disease or other pathology. It is well known that glial cells become reactive following brain or spinal cord injury, after a stroke or neurodegenerative diseases such as Alzheimer’s disease. These reactive glial cells can proliferate and maintain a high number in the injury site, and eventually form a dense scar tissue called glial scar to prevent the growth of neurons. There is an urgent need for methods that can replenish neurons in a subject, who for example suffers from an injury or pathological condition in the nervous system. The present disclosure meets this need.

3. SUMMARY

Provided herein are lipid nanoparticle compositions that can be used to convert glial cells into functional neuron. In one aspect, provided herein is a lipid nanoparticle composition comprising a nucleic acid molecule and at least one lipid, wherein the nucleic acid molecule comprises an expression sequence encoding a NeuroD1 polypeptide; and wherein the at least one lipid forms lipid nanoparticles encompassing the nucleic acid molecule.

In some embodiments, the expression sequence is a multi-cistronic sequence encoding the NeuroD1 polypeptide and at least one second polypeptide selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof.

In some embodiments, the at least one second polypeptide comprises two polypeptides each independently selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, and Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof.

In some embodiments, the expression sequence encodes (a) the NeuroD1 polypeptide alone. In alternative embodiments, the expression sequence encodes (b) the NeuroD1 polypeptide and the Dlx2 polypeptide. In alternative embodiments, the expression sequence encodes (c) the NeuroD1 polypeptide and the Isl1 polypeptide. In alternative embodiments, the expression sequence encodes (d) the NeuroD1 polypeptide and the Ascl1 polypeptide. In alternative embodiments, the expression sequence encodes (e) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Isl1 polypeptide. In alternative embodiments, the expression sequence encodes (f) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ascl1 polypeptide. In alternative embodiments, the expression sequence encodes (g) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ngn2 polypeptide. In alternative embodiments, the expression sequence encodes (h) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ctip2 polypeptide. In alternative embodiments, the expression sequence encodes (i) the NeuroD1 polypeptide, the Isl1 polypeptide, and the Ascl1 polypeptide. In alternative embodiments, the expression sequence encodes (j) the NeuroD1 polypeptide, the Ils1 polypeptide, and the Lhx3 polypeptide. In alternative embodiments, the expression sequence encodes (k) the NeuroD1 polypeptide, the Ascl1 polypeptide, and the Ctip2 polypeptide. In alternative embodiments, the expression sequence encodes (l) the NeuroD1 polypeptide, the Dlx2 polypeptide, the Ascl1 polypeptide, and the Isl1 polypeptide. In alternative embodiments, the expression sequence encodes (m) the NeuroD1 polypeptide, the Dlx2 polypeptide, the Ascl1 polypeptide, and the Ctip2 polypeptide.

In some embodiments, the expression sequence comprises at least one ribosomal skipping element located between sequences encoding the NeuroD1 polypeptide and the at least one second polypeptide. In some embodiments, the ribosomal skipping element encodes a proteasome cleavage site selected from thosea-asigna virus 2A peptide (T2A) , porcine teschovirus-1 2 A peptide (P2A) , foot-and-mouth disease virus 2 A peptide (F2A) , equine rhinitis A vims 2A peptide (E2A) , cytoplasmic polyhedrosis vims 2A peptide (BmCPV 2A) , or flacherie vims of B. mori 2A peptide (BmIFV 2A) .

In some embodiments, the nucleic acid molecule is a linear mRNA molecule, and wherein the expression sequence comprises one or more open reading frames (ORFs) , and wherein at least one of the ORFs encodes the NeuroD1 polypeptide.

In some embodiments, the expression sequence further comprises a 5’ untranslated region (5’-UTR) upstream to the open reading frame and/or a 3’-UTR downstream of the open reading frame. In some embodiments, the 5’-UTR comprises an IRES. In some embodiments, the 5’-UTR comprises the sequence selected from the group consisting of SEQ ID NOS: 53 and 55. In some embodiments, the 3’-UTR comprises the sequence selected from the group consisting of SEQ ID NOS: 57 and 59. In some embodiments, the 5’-UTR comprises a 5’-cap structure. In some embodiments, the 3’-UTR further comprises a polyA region of about 60 to about 200 residues in length.

In some embodiments, the encoded NeuroD1 polypeptide is human NeuroD1 or a functional derivative thereof. In some embodiments, the functional variant of human NeuroD1 comprises an amino acid sequence that has at least about 90%, at least about 95%, at least about 97%, or at least about 99%sequence identity to SEQ ID NO: 1. In some embodiments, the functional variant of human NeuroD1 comprises an amino acid sequence that has at least about 90%, at least about 95%, at least about 97%, or at least about 99%sequence identity to SEQ ID NO: 2.

In some embodiments, the expression sequence comprises a coding sequence for the NeuroD1 polypeptide having:

(a) the DNA sequence selected from SEQ ID NOS: 12 to 14,

(b) a codon-optimized variant of (a) , or

(c) a transcribed RNA sequence of (a) or (b) .

In some embodiments, the expression sequence comprises a coding sequence for the NeuroD1 peptide having the RNA sequence selected from SEQ ID NOS: 15 to 17.

In some embodiments, the lipid nanoparticles comprises (a) a cationic lipid, (b) a steroid, (c) a phospholipid, and (d) a polymer conjugated lipid.

In one aspect, provided herein is a linear precursor RNA molecule comprising a central region, wherein the central region comprises in the following order:

(a) a 3’ group I intron fragment or an analog thereof;

(b) an IRES;

(c) an expression sequence encoding one or more polypeptide comprising a NeuroD1 polypeptide, and

(d) a 5’ group I intron fragment or an analog thereof;

wherein the linear precursor RNA is capable of self-splicing into a circular RNA.

In some embodiments, the linear precursor RNA molecule further comprises a pair of internal complementary sequences configured to form a second double stranded region by complementary base-pairing under a suitable hybridization condition, and wherein one of the pair of internal complementary sequences is located between elements (a) and (b) and the other of the pair of internal complementary sequences is located between elements (c) and (d) , respectively. In some embodiments, wherein the pair of internal complementary sequences have about 85%to about 100%complementarity when read in the opposite directions of one another.

In some embodiments, the linear precursor RNA molecule further comprising at least one internal spacer sequence located between elements (a) and (d) . In some embodiments, the at least one internal spacer sequence comprises two internal spacer sequences located between elements (a) and (b) and between elements (c) and (d) , respectively. In some embodiments, the internal spacer is about 10 to about 80 nucleotides long.

In one aspect, provided herein is a circular RNA molecule formed by circulation of the linear precursor RNA molecules as described herein through a ribozyme self-splicing reaction of the linear precursor RNA molecule.

In one aspect, provided herein is a linear precursor RNA comprising the sequence of SEQ ID NO:66 or 68. In one aspect, provided herein is a circular RNA molecule comprising the sequence of SEQ ID NO: 67 or 69.

In one aspect, provided herein is a circular RNA molecule comprising, in the following order:

(a) a post-splicing 3’ group I intron fragment;

(b) an IRES;

(d) a post-splicing 5’ group I intron fragment.

In some embodiments, the post-splicing 3’ group I intron fragment comprises the sequence of SEQ ID NO: 64. In some embodiments, the post-splicing 5’ group I intron fragment comprises the sequence of SEQ ID NO: 65.

In some embodiments, the circular RNA molecule further comprises at least one internal spacer sequence located between elements (a) and (d) . In some embodiments, the at least one internal spacer comprises two internal spacer sequences located between elements (a) and (b) and between elements (c) and (d) , respectively. In some embodiments, the internal spacer is about 10 to about 80 nucleotides long.

In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence is a multi-cistronic sequence encoding the NeuroD1 polypeptide and at least one second polypeptide selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof.

In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the at least one second polypeptide comprises two polypeptides each independently selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof.

In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence encodes (a) the NeuroD1 polypeptide. In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence encodes (b) the NeuroD1 polypeptide and the Dlx2 polypeptide. In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence encodes (c) the NeuroD1 polypeptide and the Isl1 polypeptide. In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence encodes (d) the NeuroD1 polypeptide and the Ascl1 polypeptide. In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence encodes (e) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Isl1 polypeptide. In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence encodes (f) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ascl1 polypeptide. In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence encodes (g) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ngn2 polypeptide. In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence encodes (h) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ctip2 polypeptide. In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence encodes (i) the NeuroD1 polypeptide, the Isl1 polypeptide, and the Ascl1 polypeptide. In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence encodes (j) the NeuroD1 polypeptide, the Ils1 polypeptide, and the Lhx3 polypeptide. In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence encodes (k) the NeuroD1 polypeptide, the Ascl1 polypeptide, and the Ctip2 polypeptide. In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence encodes (l) the NeuroD1 polypeptide, the Dlx2 polypeptide, the Ascl1 polypeptide, and the Isl1 polypeptide. In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence encodes (m) the NeuroD1 polypeptide, the Dlx2 polypeptide, the Ascl1 polypeptide, and the Ctip2 polypeptide.

In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence comprises at least one ribosomal skipping element located between sequences encoding the NeuroD1 polypeptide and the at least one second polypeptide.

In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the ribosomal skipping element encodes a proteasome cleavage site selected from thosea-asigna virus 2A peptide (T2A) , porcine teschovirus-1 2 A peptide (P2A) , foot-and-mouth disease virus 2 A peptide (F2A) , equine rhinitis A vims 2A peptide (E2A) , cytoplasmic polyhedrosis vims 2A peptide (BmCPV 2A) , or flacherie vims of B. mori 2A peptide (BmIFV 2A) .

In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the encoded NeuroD1 polypeptide is human NeuroD1 or a functional variant thereof. In some embodiments, the functional variant of human NeuroD1 comprises an amino acid sequence that at least about 90%, at least about 95%, at least about 97%, or at least about 99%sequence identity to SEQ ID NO: 1. In some embodiments, the functional variant of human NeuroD1 comprises an amino acid sequence that at least about 90%, at least about 95%, at least about 97%, or at least about 99%sequence identity to SEQ ID NO: 2.

In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence comprises a coding sequence for the NeuroD1 polypeptide having:

(a) the DNA sequence selected from SEQ ID NOS: 12 to 14,

(b) a codon-optimized variant of (a) , or

(c) a transcribed RNA sequence of (a) or (b) .

In some embodiments of the linear precursor RNA molecule or circular RNA molecule described herein, the expression sequence comprises a coding sequence for the NeuroD1 peptide having the RNA sequence selected from SEQ ID NOS: 15 to 17.

In one aspect, provided herein is a lipid nanoparticle composition comprising (a) cationic lipid, (b) a steroid, (c) a phospholipid, (d) a polymer conjugated lipid, and (e) one or more of the linear precursor RNA molecule or circular RNA molecule as described herein.

In one aspect, provided herein is a composition comprising a plurality of species of nucleic acid molecules each comprising an expression sequence, wherein each expression sequence encodes one or more polypeptide comprising a NeuroD1 polynucleotide, and wherein the one or more polypeptides encoded by at least two species of the plurality of species of nucleic acid molecules are different.

In some embodiments, the one or more polypeptide encoded by at least one species of the plurality of species of nucleic acid molecules further comprises at least one second polypeptide selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof.

In some embodiments, the one or more polypeptide encoded by at least one species of the plurality of species of nucleic acid molecules further comprises at least two second polypeptides selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof.

In some embodiments, the at least one species of the plurality of species of nucleic acid molecules comprises a ribosomal skipping element between the sequence encoding NeuroD1 and the sequence encoding the at least one second polypeptide selected from selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof.

In some embodiments, the ribosomal skipping element encodes a cleavable fragment selected from T2A, P2A, F2A, E2A, BmCPV 2A and BmIFV 2A.

In some embodiments, one or more polypeptides encoded by the plurality of species of nucleic acid molecules comprise the polypeptide or combination of polypeptides selected from any one of

(a) to (m) :

(a) the NeuroD1 polypeptide;

(b) the NeuroD1 polypeptide and the Dlx2 polypeptide;

(c) the NeuroD1 polypeptide and the Isl1 polypeptide;

(d) the NeuroD1 polypeptide and the Ascl1 polypeptide;

(e) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Isl1 polypeptide;

(f) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ascl1 polypeptide;

(g) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ngn2 polypeptide;

(h) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ctip2 polypeptide;

(i) the NeuroD1 polypeptide, the Isl1 polypeptide, and the Ascl1 polypeptide;

(j) the NeuroD1 polypeptide, the Ils1 polypeptide, and the Lhx3 polypeptide;

(k) the NeuroD1 polypeptide, the Ascl1 polypeptide, and the Ctip2 polypeptide;

(l) the NeuroD1 polypeptide, the Dlx2 polypeptide, the Ascl1 polypeptide, and the Isl1 polypeptide; or

(m) the NeuroD1 polypeptide, the Dlx2 polypeptide, the Ascl1 polypeptide, and the Ctip2 polypeptide.

In some embodiments, the NeuroD1 is human NeuroD1 or a functional variant thereof. In some embodiments, the functional variant of human NeuroD1 comprises an amino acid sequence that has at least about 90%, at least about 95%, at least about 97%, or at least about 99%sequence identity to SEQ ID NO: 1. In some embodiments, the functional variant of human NeuroD1 comprises an amino acid sequence that has at least about 90%, at least about 95%, at least about 97%, or at least about 99%sequence identity to SEQ ID NO: 2.

(a) the DNA sequence selected from SEQ ID NOS: 12 to 14,

(b) a codon-optimized variant of (a) , or

(c) a transcribed RNA sequence of (a) or (b) .

In some embodiments, the plurality of species of nucleic acid molecules comprise at least one linear mRNA molecule. In some embodiments, the linear mRNA molecule further comprises a 3’-UTR and/or a 5’-UTR; optionally wherein the 5’-UTR comprises the sequence selected from the group consisting of SEQ ID NOS: 53 and 55; optionally wherein the 3’-UTR comprises the sequence selected from the group consisting of SEQ ID NOS: 57 and 59.

In some embodiments, the plurality of species of nucleic acid molecules comprise at least one circular RNA molecule. In some embodiments, the circular RNA molecule comprises, in the following order:

(a) a post-slicing 3’ group I intron fragment or an analog thereof; optionally the post-slicing 3’ group I intron fragment comprises the sequence of SEQ ID NO: 64;

(b) an IRES;

(d) a post-slicing 5’ group I intron fragment or an analog thereof; optionally the post-slicing 5’ group I intron fragment comprises the sequence of SEQ ID NO: 65.

In some embodiments, the circular RNA molecule further comprises at least one pair of internal complementary sequences configured to form a double stranded region by complementary base-pairing under a suitable hybridization condition, wherein the at least one pair of internal complementary sequences are both located between elements (a) and (d) . In some embodiments, one of the pair of internal complementary sequences is located between elements (a) and (b) and the other of the pair of internal complementary sequences is located between elements (c) and (d) , respectively. In some embodiments, the pair of internal complementary sequences have about 85%to about 100%complementarity with respect to one another.

In some embodiments, the plurality of species of nucleic acid molecules comprise at least one linear precursor RNA molecule, wherein the linear precursor RNA molecule comprises a central region, wherein the central region comprises in the following order:

(a) a 3’ group I intron fragment or an analog thereof;

(b) an IRES;

(d) a 5’ group I intron fragment or an analog thereof.

In some embodiments, the linear precursor RNA molecule further comprises at least one internal spacer sequence located between elements (a) and (d) . In some embodiments, the at least one internal spacer sequence comprises two internal spacer sequences located between elements (a) and (b) and between elements (c) and (d) , respectively.

In some embodiments, the composition further comprises at least one lipid. In some embodiments, the at least one lipid forms lipid nanoparticles encompassing one or more species of the plurality of species of nucleic acid molecules. In some embodiments, the lipid nanoparticle comprises (a) a cationic lipid, (b) a steroid, (c) a phospholipid, and (d) a polymer conjugated lipid. In some embodiments, the composition is a lipid nanoparticle composition.

In one aspect, provided herein is a method of converting a starting population of glial cells into functional neurons, comprising contacting the starting population of glial cells with the lipid nanoparticle composition as provided herein under a suitable condition, wherein upon the contacting, at least one glial cell in the starting population trans-differentiates into a functional neuron.

In one aspect, provided herein is a method of converting a starting population of glial cells into functional neurons, comprising contacting the starting population of glial cells with the linear precursor RNA molecule or the circular RNA molecule as provided herein under a suitable condition, wherein upon the contacting, at least one glial cell in the starting population trans-differentiates into a functional neuron.

In one aspect, provided herein is a method of converting a starting population of glial cells into functional neurons, comprising contacting the starting population of glial cells with the composition comprising the plurality of species of nucleic acid molecules as provided herein under a suitable condition, wherein upon the contacting, at least one glial cell in the starting population trans-differentiates into a functional neuron.

In some embodiments, the starting population of glial cells comprises astrocytes, NG2 cells, Müller cells, and/or microglia cells.

In some embodiments, the functional neuron has at least one neuronal phenotype selected from neuronal morphology, expression of one or more neuronal marker, lack of expression of one or more glial cell marker, electrophysiologic characteristics of neurons, exon, dendrite and/or synapse formation, and release of neurotransmitters.

In some embodiments, the functional neuron expresses one or more neuronal markers selected from doublecortin (DCX) , class III beta tubulin (TUJ1) , neuronal specific nuclear protein (NeuN) , microtubule associated protein 2 (Map2) , RNA Binding Protein, MRNA Processing Factor (Rbpms) , brain-specific homeobox/POU domain protein 3A (Brn3a) , and Opsins.

In some embodiments, the trans-differentiated functional neuron stops expressing one or more glial cell marker selected from glia fibrillary acidic protein (GFAP) , aldehyde dehydrogenase 1 family, member L1 (AldhlL1) , S100 calcium-binding protein B (S100β) , SRY-box transcription factor 9 (Sox9) .

In some embodiments, the functional neuron is capable of firing action potentials.

In some embodiments, the functional neuron releases neurotransmitters selected from glutamate, GABA, dopamine, glycine, serotonin, and noradrenaline.

In some embodiments, the starting population of glial cells are in an in vitro cell culture.

In some embodiments, the starting population of glial cells are located in situ in a subject. In some embodiments, the glial cells are located in the brain or spinal cord of the subject. In some embodiments, the glial cells are located in the peripheral nervous system of the subject. In some embodiments, the glial cells are located in the eye of the subject. In some embodiments, the glial cells are located in the striatum of the brain.

In some embodiments, the contacting is performed by administration to the subject an effective amount of the lipid nanoparticle composition as described herein. In some embodiments, the contacting is performed by administration to the subject an effective amount of the linear precursor RNA molecule or the circular RNA molecule as described herein. In some embodiments, the contacting is performed by administration to the subject an effective amount of the composition comprising the plurality of species of nucleic acid molecules as described herein.

In one aspect, provided herein is a method of producing a neuronal phenotype is a glial cell comprising contacting the glial cells with (a) the lipid nanoparticle composition as described herein, (b) the linear precursor RNA molecule or the circular RNA molecule as described herein, and/or (c) the composition comprising the plurality of species of nucleic acid molecules as described herein under a suitable condition, wherein upon the contacting, the glial cell produces a detectable neuronal phenotype.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the expression of the neural transcription factor NeuroD1 (red) in rat primary astrocytes 24 hours post transfection of the NeuroD1-encoding mRNA. Astrocytes were labeled with green staining of glia fibrillary acidic protein (GFAP) and all cell nuclei are label with DAPI (blue) .

FIG. 2 shows neurons in red (NeuN+) converted from rat primary astrocytes 21 days post transfection of mRNA encoding NeuroD1. Astrocytes were shown in green (GFAP+) and all cell nuclei were labeled with DAPI (blue) .

FIG. 3 shows the expression of NeuroD1 (red) in astrocytes 24 hours post transfection of the NeuroD1-encoding cirRNA. Astrocytes are labeled with green staining of GFAP and all cell nuclei were labeled with DAPI (blue) .

FIGS. 4A and 4B show astrocytes were converted into neurons after transfection with mRNA encoding NeuroD1. Astrocytes were labeled with green (GFAP+) ; all cell nuclei were labeled with DAPI (blue) ; neuron marker TUJ1 was labeled in red.

FIGS. 5A and 5B show in vivo neural transcription factor NeuroD1 protein expression 24 hours after injection of LNP composition containing NeuroD1 encoding RNA. NeuroD1 protein expression was shown in purple, GFP protein was shown in green, astrocyte marker (GFAP+) was shown in red and all nuclei (DAPI+) were shown in blue.

FIGS. 6A and 6B show in vivo conversion of astrocytes to neurons 14 days after injection of LNP/NeuroD1-p2A-GFP-mRNA. Green marks ND1-p2A-GFP-mRNA transfected cells, astrocytes are labeled red (GFAP) , mature neurons are labeled purple (NeuN) . All nuclei (DAPI+) show in blue.

FIG. 7 shows the sequence alignment of NeuroD1 proteins from various species, including house mouse (Mus musculus) (SEQ ID NO: 73) , zebrafish Danio rerio) (SEQ ID NO: 74) , human (Homo Sapiens) (SEQ ID NO: 1) , rat (Rattus norvegicus) (SEQ ID NO: 75) , chicken (Gallus gallus) (SEQ ID NO: 76) , cattle (Bos taurus) (SEQ ID NO: 77) , hamster (Mesocricetus auratus) (SEQ ID NO: 78) , pig (Sus scrofa) (SEQ ID NO: 79) , frog (Xenopus tropicalis) (SEQ ID NO: 80) , dog (Canis lupus familiaris) (SEQ ID NO: 81) , sheep (Ovis aries) (SEQ ID NO: 82) , and chimpanzee (Pan troglodytes) (SEQ ID NO: 83) .

FIG. 8 shows the expression of the neural transcription factor NeuroD1 (left panel, red) and Ascl1 (middle panel, green) in rat primary astrocytes 4 hours post co-transfection of the NeuroD1-encoding mRNA and the Ascl1-encoding mRNA. DAPI (4', 6-diamidino-2-phenylindole) stained all cell nuclei in blue (right panel, all color) .

FIG. 9 shows neurons generated by trans-differentiation of rat primary astrocytes. The generated neurons showed NeuN-positive (left panel, red) and MAP2-positive (middle panel, green) staining 2 weeks after co-transfection of the rat primary astrocytes with the NeuroD1-encoding mRNA and the Ascl1-encoding mRNA. DAPI stained all cell nuclei in blue (right panel, all color) .

FIG. 10 shows neurons generated by trans-differentiation of rat primary astrocytes. The generated neurons showed NeuN-positive (left panel, red) and MAP2-positive (middle panel, green) .

FIG. 11 shows the expression of the neural transcription factor Dlx2 (left panel, red) and NeuroD1 (middle panel, green) in rat primary astrocytes 4 hours post co-transfection of the NeuroD1-encoding mRNA and the Dlx2-encoding mRNA. DAPI (4', 6-diamidino-2-phenylindole) stained all cell nuclei in blue (right panel, all color) .

FIG. 12 shows neurons generated by trans-differentiation of rat primary astrocytes. The generated neurons showed NeuN-positive (left panel, red) and MAP2-positive (middle panel, green) staining 2 weeks after co-transfection of the rat primary astrocytes with the NeuroD1-encoding mRNA and the Dlx2-encoding mRNA. DAPI stained all cell nuclei in blue (right panel, all color) .

FIG. 13 shows neurons generated by trans-differentiation of rat primary astrocytes. The generated neurons showed NeuN-positive (left panel, red) and MAP2-positive (middle panel, green) staining 3 weeks after co-transfection of the rat primary astrocytes with the NeuroD1-encoding mRNA and the Dlx2-encoding mRNA. DAPI stained all cell nuclei in blue (right panel, all color) .

FIG. 14 shows some of the neurons generated by trans-differentiation of rat primary astrocytes were glutamatergic neurons. Particularly, the generated neurons showed vGlut1-positive (left panel, red) and MAP2-positive (middle panel, green) staining 3 weeks after co-transfection of the rat primary astrocytes with the NeuroD1-encoding mRNA and the Dlx2-encoding mRNA. DAPI stained all cell nuclei in blue (right panel, all color) . The vGlut1 staining was a maker for glutamatergic neurons.

FIG. 15 shows some of the neurons generated by trans-differentiation of rat primary astrocytes were GABAergic neurons. Particularly, the generated neurons showed GABA-positive (left panel, red) and MAP2-positive (middle panel, green) staining 3 weeks after co-transfection of the rat primary astrocytes with the NeuroD1-encoding mRNA and the Dlx2-encoding mRNA. DAPI stained all cell nuclei in blue (right panel, all color) . The GABA staining was a maker for GABAergic neurons.

FIG. 16 shows the expression of the neural transcription factor Dlx2 (left panel, red) and Ascl1 (middle panel, green) in rat primary astrocytes 4 hours post co-transfection of the Ascl1-encoding mRNA and the Dlx2-encoding mRNA. DAPI (4', 6-diamidino-2-phenylindole) stained all cell nuclei in blue (right panel, all color) .

FIG. 17 shows neurons generated by trans-differentiation of rat primary astrocytes. The generated neurons showed NeuN-positive (left panel, red) and MAP2-positive (middle panel, green) staining 2 weeks after co-transfection of the rat primary astrocytes with the Ascl1-encoding mRNA and the Dlx2-encoding mRNA. DAPI stained all cell nuclei in blue (right panel, all color) .

5. DETAILED DESCRIPTION

Provided herein are methods and compositions for producing new neurons from glial cells both in vitro and in vivo. Aspects of the present invention include introducing NeuroD1 alone or in combination with one or more protein factors into a glial cell, particularly into a astrocyte, a reactive astrocyte, a NG2 cell, a reactive NG2 cell, a microglial cell or a Müller cell, thereby converting the reactive glial cell to a functional neuron. Related methods of producing a neuronal phenotype in a glial cell are also provided according to aspects of the present invention which include expressing NeuroDl alone or in combination with one or more protein factors in the glial cell, wherein expressing NeuroDl includes delivering one or more nucleic acid molecules, particularly RNA molecules, formulated as part of lipid nanoparticles to the glial cell. Additional features of the present disclosure will become apparent to those skilled in the art upon consideration of the following detailed description of particular embodiments.

5.1 General Techniques

Techniques and procedures described or referenced herein include those that are generally well understood and/or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed. 2001) ; Current Protocols in Molecular Biology (Ausubel et al. eds., 2003) .

5.2 Terminology

Unless described otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of term set forth below shall control.

The term “polynucleotide” or “nucleic acid, ” as used interchangeably herein, refers to polymers of nucleotides of any length and includes, e.g., DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. Nucleic acid can be in either single-or double-stranded forms. As used herein and unless otherwise specified, “nucleic acid” also includes nucleic acid mimics such as locked nucleic acids (LNAs) , peptide nucleic acids (PNAs) , and morpholinos. “Oligonucleotide, ” as used herein, refers to short synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides. Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences” ; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences. ”

As used herein, the term “wild-type” refers to organisms, cells, genes, proteins, oligonucleotides, and the like that are found in Nature and are unchanged relative to these components found in Nature (native or in the wild) .

As used herein, the term “non-naturally occurring” when used in reference to a nucleic acid molecule as described herein is intended to mean that the nucleic acid molecule is not found in nature. A non-naturally occurring nucleic acid encoding a protein (e.g., NeuroD1) contains at least one genetic alternation or chemical modification not normally found in a naturally occurring nucleic acid, including a wild-type nucleic acid. Genetic alterations include, for example, modifications to an expressible nucleic acid sequences encoding heterologous peptides or polypeptides, other nucleic acid additions, nucleic acid deletions, nucleic acid substitution, and/or other functional disruption of a coding sequence. Such modifications include, for example, modifications in the coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides. Additional modifications include, for example, modifications in non-coding regulatory regions in which the modifications alter expression of a gene or operon. Additional modifications also include, for example, incorporation of a nucleic acid sequence into a vector, such as a plasmid or an artificial chromosome. Chemical modifications include, for example, one or more functional nucleotide analog as described herein.

An “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA, or a mixed nucleic acids, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an “isolated” nucleic acid molecule, such as an mRNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding a polypeptide as described herein are isolated or purified. The term embraces nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA or RNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure molecule may include isolated forms of the molecule.

The term “encoding nucleic acid” or grammatical equivalents thereof as it is used in reference to nucleic acid molecule encompasses (a) a nucleic acid molecule in its native state or when manipulated by methods well known to those skilled in the art that can be transcribed to produce mRNA which is then translated into a peptide and/or polypeptide, and (b) the mRNA molecule itself. The antisense strand is the complement of such a nucleic acid molecule, and the encoding sequence can be deduced therefrom. The term “coding region” refers to a portion in an encoding nucleic acid sequence that is translated into a peptide or polypeptide. The term “untranslated region” or “UTR” refers to the portion of an encoding nucleic acid that is not translated into a peptide or polypeptide. Depending on the orientation of a UTR with respect to the coding region of a nucleic acid molecule, a UTR is referred to as the 5’-UTR if located to the 5’-end of a coding region, and a UTR is referred to as the 3’-UTR if located to the 3’-end of a coding region.

An encoding nucleic acid can be mono-cistronic or multi-cistronic. A “mono-cistronic sequence” refers to a polynucleotide that comprises coding sequence for a single peptide or polypeptide chain. A “multi-cistronic sequence” refers to a polynucleotide that comprises coding sequences for two or more peptide and/or polypeptide chains.

The term “mRNA” as used herein refers to a message RNA molecule comprising one or more open reading frame (ORF) that can be translated by a cell or an organism provided with the mRNA to produce one or more peptide or protein product. The region containing the one or more ORFs is referred to as the coding region of the mRNA molecule. In certain embodiments, the mRNA molecule further comprises one or more untranslated regions (UTRs) . In certain embodiments, the mRNA is or is part of a linear RNA molecule. In other embodiments, the mRNA is or is part of a circular RNA molecule.

In certain embodiments, the mRNA is a monocistronic mRNA that comprises only one ORF. In certain embodiments, the monocistronic mRNA encodes a peptide or protein comprising at least one epitope of a selected polypeptide (e.g., transcription factor) . In other embodiments, the mRNA is a multicistronic mRNA that comprises two or more ORFs. In certain embodiments, the multiecistronic mRNA encodes two or more peptides or proteins that can be the same or different from each other.

As used herein, the term “ribosomal skipping element” refers to a nucleotide sequence capable of causing generation of two polypeptide chains from translation of one RNA molecule. In some embodiments, the ribosomal skipping element can terminate translation of the first polypeptide chain and re-initiating translation of the second polypeptide chain from the RNA molecule. In alternative embodiments, the ribosomal skipping element encodes a protease cleavage site in the polypeptide encoded by the RNA molecule, so that the polypeptide can be cleaved by an intrinsic protease activity of its own, or by another protease in its environment to produce two polypeptide chains. In specific embodiments, the ribosomal skipping element encodes thosea-asigna virus 2A peptide (T2A) , porcine teschovirus-1 2 A peptide (P2A) , foot-and-mouth disease virus 2 A peptide (F2A) , equine rhinitis A vims 2A peptide (E2A) , cytoplasmic polyhedrosis vims 2A peptide (BmCPV 2A) , or flacherie vims of B. mori 2A peptide (BmIFV 2A) .

The term “nucleobases” encompasses purines and pyrimidines, including natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural or synthetic analogs or derivatives thereof.

The term “functional nucleotide analog” as used herein refers to a modified version of a canonical nucleotide A, G, C, U or T that (a) retains the base-pairing properties of the corresponding canonical nucleotide, and (b) contains at least one chemical modification to (i) the nucleobase, (ii) the sugar group, (iii) the phosphate group, or (iv) any combinations of (i) to (iii) , of the corresponding natural nucleotide. As used herein, “base pairing” encompasses not only the canonical Watson-Crick adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between canonical nucleotides and functional nucleotide analogs or between a pair of functional nucleotide analogs, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a modified nucleobase and a canonical nucleobase or between two complementary modified nucleobase structures. For example, a functional analog of guanosine (G) retains the ability to base-pair with cytosine (C) or a functional analog of cytosine. One example of such non-canonical base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. As described herein, a functional nucleotide analog can be either naturally occurring or non-naturally occurring. Accordingly, a nucleic acid molecule containing a functional nucleotide analog can have at least one modified nucleobase, sugar group and/or internucleoside linkage. Exemplary chemical modifications to the nucleobases, sugar groups, or internucleoside linkages of a nucleic acid molecule are provided herein.

The terms “complement” or “complementary” can be determined by the Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a “percent complementarity” to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3’-TCGA-5’ is 100%complementary to the nucleotide sequence 5’-AGCT-3’. Further, the nucleotide sequence 3’-TCGA-is 100%complementary to a region of the nucleotide sequence 5’-TTAGCTGG-3’.

The terms “duplexed, ” “double-stranded, ” or “hybridized” as used herein refer to multiple nucleic acid molecules or a region of a single nucleic acid molecue (e.g., the stem region in a stem-loop structure) that is formed by hybridization of two single strands of nucleic acids containing complementary sequences. As described herein, a pair of complementary sequences can be fully complementary or partially complementary.

The terms “hybridization” and “hybridizes” refer to pairing and binding of complementary nucleic acids. Hybridization occurs to varying extents between two nucleic acids depending on factors such as the degree of complementarity of the nucleic acids, the melting temperature, Tm, of the nucleic acids and the stringency of hybridization conditions, as is well known in the art. The term “stringency of hybridization conditions” refers to conditions of temperature, ionic strength, and composition of a hybridization medium with respect to particular common additives such as formamide and Denhardt's solution. Determination of particular hybridization conditions relating to a specified nucleic acid is routine and is well known in the art, for instance, as described in J. Sambrook and D. W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; and F. M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002. High stringency hybridization conditions are those which only allow hybridization of substantially complementary nucleic acids. Typically, nucleic acids having about 85-100%complementarity are considered highly complementary and hybridize under high stringency conditions. Intermediate stringency conditions are exemplified by conditions under which nucleic acids having intermediate complementarity, about 50-84%complementarity, as well as those having a high degree of complementarity, hybridize. In contrast, low stringency hybridization conditions are those in which nucleic acids having a low degree of complementarity hybridize.

The terms “specific hybridization” and “specifically hybridizes” refer to hybridization of a particular nucleic acid to a target nucleic acid without substantial hybridization to nucleic acids other than the target nucleic acid in a sample. Stringency of hybridization and washing conditions depends on several factors, including the Tm of the probe and target and ionic strength of the hybridization and wash conditions, as is well-known to the skilled artisan. Hybridization and conditions to achieve a desired hybridization stringency are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001; and Ausubel, F. et al., (Eds. ) , Short Protocols in Molecular Biology, Wiley, 2002. An example of high stringency hybridization conditions is hybridization of nucleic acids over about 100 nucleotides in length in a solution containing 6X SSC, 5X Denhardt's solution, 30%formamide, and 100 micrograms/ml denatured salmon sperm at 37℃ overnight followed by washing in a solution of 0.1X SSC and 0.1%SDS at 60℃ for 15 minutes. SSC is 0.15M NaCl/0.015M Na citrate. Denhardt's solution is 0.02%bovine serum albumin/0.02%FICOLL/0.02%polyvinylpyrrolidone. Under highly stringent conditions, SEQ ID NO: 2 will hybridize to the complement of substantially identical targets and not to unrelated sequences.

The term “operably linked” as used herein refers to a nucleic acid sequence in functional relationship with a second nucleic acid sequence. The term “operably linked” encompasses functional connection of two or more nucleic acid sequences, such as a nucleic acid to be transcribed and a regulatory element. The term “regulatory element” as used herein refers to a nucleotide sequence which controls some aspect of the expression of an operably linked nucleic acid coding sequence. Exemplary regulatory elements include an enhancer, such as, but not limited to: woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) ; an internal ribosome entry site (IRES) or a 2A domain; an intron (e.g., a group I intron) ; an origin of replication; a polyadenylation signal (pA) ; a promoter; a transcription termination sequence; and an upstream regulatory domain, which contribute to the replication, transcription, posttranscriptional processing of an operably linked nucleic acid sequence. Those of ordinary skill in the art are capable of selecting and using these and other regulatory elements in an expression vector with no more than routine experimentation.

The terms “translational enhancer element, ” “TEE” and “translational enhancers” as used herein refers to an region in a nucleic acid molecule that functions to promotes translation of a operably linked coding sequence of the nucleic acid into a protein or peptide product, such as via cap-dependent or cap-independent translation. A TEE typically locates in the UTR region of a nucleic acid molecule (e.g., mRNA) and enhance the translational level of a coding sequence located either upstream or downstream. For example, a TEE in a 5’-UTR of a nucleic acid molecule can locate between the promoter and the starting codon of the nucleic acid molecule. Various TEE sequences are known in the art (Wellensiek et al. Genome-wide profiling of human cap-independent translation-enhancing elements, Nature Methods, 2013 Aug; 10 (8) : 747–750; Chappell et al. PNAS June 29, 2004 101 (26) 9590-9594) . Some TEEs are known to be conserved across multiple species (Pánek et al. Nucleic Acids Research, Volume 41, Issue 16, 1 September 2013, Pages 7625–7634) . In particular embodiments, a TEE is a promoter.

The term “promoter” is a term of art and is used herein to refer to a nucleic acid sequence operably linked to a nucleic acid sequence to be transcribed such as a nucleic acid sequence encoding a NeuroD1 polypeptide as described herein. In some embodiments, a promoter is positioned upstream of a nucleic acid sequence to be transcribed and provides a site for specific binding by RNA polymerase and other transcription factors. In specific embodiments, a promoter is positioned upstream of the nucleic acid sequence transcribed to produce the desired molecule, and provides a site for specific binding by RNA polymerase and other transcription factors.

In some embodiments, a promoter specifically enhances expression of an operably linked nucleic acid in a given cell type, and such promoter is referred to as a “cell type-specific promoter. ” In certain embodiments, a cell type-specific promoter is a glial cell specific promoter. Non-limiting examples of glial cell-specific promoters that can be used in connection with the present disclosure include but are not limited to glial fibrillary acidic protein (GFAP) promoter and aldehyde dehydrogenase 1 family, member L1 (AldhlL1) promoter, a lipocalin 2 (lcn2) promoter, a S100 calcium-binding protein B (S100β) promoter, a SRY-box transcription factor 9 (Sox9) promoter. A non-limiting example of an NG2 cell-specific promoter is the promoter of the chondroitin sulfate proteoglycan 4 gene, also known as neuron-glial antigen 2 (NG2) .

In alternative embodiments, a promoter generally enhances expression of an operably linked nucleic acid in various different cell types, such as at least 5 different cell types, and such promoter is referred to as an “ubiquitous promoter. ” Non-limiting examples of ubiquitous promoters that can be used in connection with the present disclosure include but are not limited to the CAG promoter which combines the cytomegalovirus CMV early enhancer element and chicken beta-actin promoter, a CMV promoter, a ubiquitin promoter, an EF-1a promoter.

As used herein, an “internal ribosome entry site” or “IRES” refers to an RNA sequence or structural element ranging in size from 10 nt to 1000 nt or more, capable of initiating translation of a polypeptide in the absence of a typical RNA cap structure. An IRES is typically about 500 nt to about 700 nt in length.

As used herein, the term “stem-loop sequence” refers to a single-stranded polynucleotide sequence having at least two regions that are complementary or substantially complementary to each other when read in opposite directions, and thus capable of base-pairing with each other to form at least one double helix and an unpaired loop. The resulting structure is known as a stem-loop structure, a hairpin, or a hairpin loop, which is a secondary structure found in many RNA molecules.

As used herein, the terms “circular RNA” refers to a polyribonucleotide that forms a circular structure through covalent bonds. According to the present disclosure, a circular RNA can be generated via any method known in the art, including but not limited to the splint-mediated method (see e.g., C.J. Kershaw, R.T. O'Keefe Methods Mol Biol. 2012; 941: 257-69) , the permuted intro-exon (PIE) method (see e.g., M. Puttaraju and Michael D. Been Nucleic Acids Res. 1992 Oct 25; 20 (20) : 5357–5364) , and the RNA ligase-mediated method.

The term “group I intron” is a term of art and refers to introns characterized by a linear array of conserved sequences and secondary structural features that are capable of autocatalyzing their removal (self-splicing) from primary transcripts by two successive transesterifications. The ability of group I introns to self-splice and therefore act as ribozymes has been well defined. Particularly, the splicing reaction is initiated with a nucleophilic attack by the 3’ hydroxyl group of guanosine (G) on the phosphorous at the 5’ splice site, resulting in cleavage of the precursor RNA. A free 3’ hydroxyl group is generated on the end of the 5’ exon, and the G is ligated to the 5’ end of the intron. Release of the intron and ligation of the exons occur in the second step by attack of the 5’ exon’s 3’ hydroxyl group on the 3’ splice site phosphorous. For a review, see e.g., Cech, T.R. and Bass, B. L. (1986) Annu. Rev. Biochem. 55, 599-629; Cech, T.R. (1990) Annu. Rev. Biochem, 59, 543-568.

As used herein, the term “splice site” refers to the dinucleotides between which cleavage of the phosphodiester bond occurs during a splicing reaction. A “5’ splice site” refers to the 5’ dinucleotides of the intron (e.g., a group I intron) , while a “3’ splice site” refers to the 3′ dinucleotide of the intron.

Without being bound by any theory, it is contemplated that the function of splice sites in a group I intron is not determined by their relative positions (5’ or 3’) , but by splice site sequence and structural features which recognize, bind, and activate the ribozyme self-splicing reaction at those sites. Thus, by reversing large sections of the intron as well as the order of the two splice sites, it is possible to utilize the entire secondary and tertiary structure of the intron to stabilize the required interactions and facilitate circular exon generation. See for example, the self-splice of the permuted intro-exon (PIE) sequence demonstrated in M. Puttaraju and Michael D. Been Nucleic Acids Res. 1992 Oct 25; 20 (20) : 5357–5364.

The term “permutation site” refers to the site in a group I intron where a cut is made to separate the intron into two functional fragments, between which the fragment containing the 5’ splice site the group I intron is herein referred to as the “5’ group I intron fragment” and the other fragment containing the 3’ splice site is herein referred to as the “3’ group I intron fragment. ” As used herein, a group I intron fragment is “functional” means the fragment retains sufficient sequence and secondary structural features of the group I intron, such that the fragment can recognize, bind, and activate the ribozyme self-splicing reaction at the splice site it contains. In some embodiments, a 5’ group I intron fragment further contains a stretch of the exon sequence adjacent to the 5’ splice site. In some embodiments, a 3’ group I intron fragment further contains a stretch of the exon sequence adjacent to the 3’ splice site.

Without being bound by any theory, it is contemplated that a 3’ group I intron fragment or a 5’ group I intron fragment can recognize, bind, and activate the ribozyme self-splicing reaction due to the formation of secondary structures by the fragment, including the formation of one or more stem-loop structures having a suitable size (e.g., stem length and loop size) at one or more suitable locations in the fragment. Accordingly, 3’ group I intron fragment or a 5’ group I intron fragment as described herein can be altered in its primary nucleic acid sequence, while retaining the function in activating the ribozyme self-splicing reaction.

The term “an analog of a 3’ group I intron fragment” refers to a sequence having at least 75%sequence identity to the 3’ group I intron fragment and is able to recognize, bind, and activate the ribozyme self-splicing reaction at the 3’ splice site it contains. In some embodiments, an analog of a 3’ group I intron fragment forms the same number of stem loop structure as the original 3’ group I intron fragment. In some embodiments, an analog of a 3’ group I intron fragment has the same number of stem loop structure as the original 3’ group I intron fragment, and each stem-loop structure having the same (a) stem length, (b) loop size, or (c) both stem length and loop size, as a corresponding stem-loop structure formed by the original 3’ group I intron fragment. In some embodiments, an analog of a 3’ group I intron fragment has the same number of stem loop structure as the original 3’ group I intron fragment, and the one or more distance between adjacent stem loop structures in the analog fragment is the same as that of the original 3’ group I intron fragment. In some embodiments, an analog of a 3’ group I intron fragment has the same number of stem loop structure as the original 3’ group I intron fragment, and each stem-loop structure having the same (a) stem length, (b) loop size, or (c) both stem length and loop size, as a corresponding stem-loop structure formed by the original 3’ group I intron fragment, and furthermore the one or more distance between adjacent stem loop structures in the analog fragment is the same as that of the original 3’ group I intron fragment.

The term “an analog of a 5’ group I intron fragment” refers to a sequence having at least 75%sequence identity to the 5’ group I intron fragment and is able to recognize, bind, and activate the ribozyme self-splicing reaction at the 5’ splice site it contains. In some embodiments, an analog of a 5’ group I intron fragment forms the same number of stem loop structure as the original 5’ group I intron fragment. In some embodiments, an analog of a 5’ group I intron fragment has the same number of stem loop structure as the original 5’ group I intron fragment, and each stem-loop structure having the same (a) stem length, (b) loop size, or (c) both stem length and loop size, as a corresponding stem-loop structure formed by the original 5’ group I intron fragment. In some embodiments, an analog of a 5’ group I intron fragment has the same number of stem loop structure as the original 5’ group I intron fragment, and the one or more distance between adjacent stem loop structures in the analog fragment is the same as that of the original 5’ group I intron fragment. In some embodiments, an analog of a 5’ group I intron fragment has the same number of stem loop structure as the original 5’ group I intron fragment, and each stem-loop structure having the same (a) stem length, (b) loop size, or (c) both stem length and loop size, as a corresponding stem-loop structure formed by the original 5’ group I intron fragment, and furthermore the one or more distance between adjacent stem loop structures in the analog fragment is the same as that of the original 5’ group I intron fragment.

In certain embodiments, a 3′ group I intron fragment is a contiguous sequence at least 75%identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical) to a 3′ proximal fragment of a natural group I intron including the 3′ splice site dinucleotide and optionally the adjacent exon sequence at least 1 nt in length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 nt in length) and at most the length of the exon. Typically, a 5′ group I intron fragment is a contiguous sequence at least 75%identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical) to a 5′ proximal fragment of a natural group I intron including the 5′ splice site dinucleotide and optionally the adjacent exon sequence at least 1 nt in length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 nt in length) and at most the length of the exon. As described by Umekage et al. (2012) , external portions of the 3′ group I intron fragment and 5′ group I intron fragment are removed in circularization, causing the circular RNA provided herein to comprise only the portion of the 3′ group I intron fragment formed by the optional exon sequence of at least 1 nt in length and 5′ group I intron fragment formed by the optional exon sequence of at least 1 nt in length, if such sequences were present on the non-circularized precursor RNA. The part of the 3′ group I intron fragment that is retained by a circular RNA is referred to herein as the post splicing 3′ group I intron fragment. The part of the 5′ group I intron fragment that is retained by a circular RNA is referred to herein as the post splicing 5′ group I intron fragment.

The term “spacer” refers to a region of a polynucleotide sequence ranging from 1 nucleotide to hundreds or thousands of nucleotides separating two other elements along a polynucleotide sequence. The sequences can be defined or can be random. A spacer is typically non-coding. In some embodiments, spacers include duplex forming regions.

Codon substitution or codon replacement in the context of codon optimization refer to replacing a codon present in a candidate nucleotide sequence (e.g., an mRNA encoding a therapeutic agent) with another codon. Thus, a codon can be substituted in a candidate nucleic acid sequence, for example, via chemical peptide synthesis or through recombinant methods known in the art. Accordingly, references to a “substitution” or “replacement” at a certain location in a nucleic acid sequence (e.g., an mRNA) or within a certain region or subsequence of a nucleic acid sequence (e.g., an mRNA) refer to the substitution of a codon at such location or region with an alternative codon. As used herein, the term “codon-optimized variant” refers to a synonymous nucleotide sequence that encodes the same polypeptide sequence encoded by a candidate nucleotide sequence (e.g., a nucleotide sequence encoding a NeuroD1 polypeptide) . Thus, there are no amino acid substitutions in the polypeptide encoded by the codon optimized nucleotide sequence with respect to the polypeptide encoded by the candidate nucleotide sequence. A candidate nucleic acid sequence can be codon-optimized by replacing all or part of its codons according to a substitution table map. According to the present disclosure, a candidate nucleotide sequence can be codon-optimized, for example, to improve its translation efficacy of the encoded polypeptide. In some embodiments, the candidate nucleotide sequence is codon-optimized for improved translation efficacy after in vivo administration, e.g., administration in a lipid nanoparticle formulation.

The term “peptide” as used herein refers to a polymer containing between two and fifty (2-50) amino acid residues linked by one or more covalent peptide bond (s) . The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid (e.g., an amino acid analog or non-natural amino acid) .

The terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of greater than fifty (50) amino acid residues linked by covalent peptide bonds. That is, a description directed to a polypeptide applies equally to a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid (e.g., an amino acid analog) . As used herein, the terms encompass amino acid chains of any length, including full length proteins (e.g., NeuroD1) .

As used herein, the term “NeuroD1 polypeptide” refers to NeuroD1 or a functional derivative of NeuroD1. The term “neurogenic differentiation 1 protein” or “NeuroD1” as used herein, refers to any native NeuroD1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated. The term encompasses unprocessed NeuroD1 as well as any form of NeuroD1 that results from processing in the cell. The term also encompasses naturally occurring variants of NeuroD1, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human NeuroD1 is MTKSYSESGLMGEPQPQGPPSWTDECLSSQDEEHEADKKEDDLETMNAEEDSLRNGGEEEDEDEDLEEEEEEEEEDDDQKPKRRGPKKKKMTKARLERFKLRRMKANARERNRMHGLNAALDNLRKVVPCYSKTQKLSKIETLRLAKNYIWALSEILRSGKSPDLVSFVQTLCKGLSQPTTNLVAGCLQLNPRTFLPEQNQDMPPHLPTASASFPVHPYSYQSPGLPSPPYGTMDSSHVFHVKPPPHAYSAALEPFFESPLTDCTSPSFDGPLSPPLSINGNFSFKHEPSAEFEKNYAFTMHYPAATLAGAQSHGSIFSGTAAPRCEIPIDNIMSFDSHSHHERVMSAQLNAIFHD (SEQ ID NO: 1; GenBank Accession NP_002491.3) . A “full-length” NeuroD1 as used herein refers to the mature, natural length NeuroD1 molecule. For example, full-length human NeuroD1 refers to a molecule that has 356 amino acids (see e.g., SEQ ID NO: 1) .

An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse NeuroD1 and human NeuroD1 can be considered orthologs for the biological function of regulating neuronal differentiation and neurogenesis. See e.g., Cho, J.H. et al., Mol, Neurobiol., 30: 35-47, 2004; Kuwabara, T. et al., Nature Neurosci., 12: 1097-1105, 2009; and Gao, Z. et al., Nature Neurosci., 12: 1090-1092, 2009. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25%to 100%amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less than 25%can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. Those skilled in the art will understand how to identify orthologous genes harboring a biological function of interest. For example, a list of orthologous NeuroD1 genes and encoded NeuroD1 protein sequences can be found on GenBank website: www. ncbi. nlm. nih. gov/gene/4760/ortholog/? scope=89593&term=NEUROD1. Non-exhaustive examples of NeuroD1 proteins from various non-human organisms as identified by their respective GenBank accession numbers include Mus musculus (house mouse) NP_035024.1, Danio rerio (zebrafish) NP_571053.1, Gallus gallus (chicken) NP_990251.2, Bos taurus (cattle) NP_001096758.1, Mesocricetus auratus (golden hamster) XP_005065174.1, Sus scrofa (pig) XP_020931169.1, Xenopus tropicalis (frog) NP_001090868.1, Canis lupus familiaris (dog) XP_005640434.2, Pan troglodytes (chimpanzee) XP_001158946.1, Ovis aries (sheep) XP_011987527.1. In some embodiments, A group orthologs genes encode protein products that can be considered functional derivatives of one another.

NeuroD1 is highly conserved in the vertebrate family. Figure 7 shows the sequence alignment of NeuroD1 proteins from various species, including mouse, zebrafish, human, rat, chicken, cattle, hamster, pig, frog, dog, chimpanzee and sheep. As shown, at least 95%amino acid residues in the NeuroD1 sequences are conserved across NeuroD1 orthologs from various species.

As used herein, the term “Dlx2 polypeptide” refers to Dlx2 or a functional derivative of Dlx2. The term “distal-less homeobox 2” or “Dlx2” as used herein, refers to any native Dlx2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated. The term encompasses unprocessed Dlx2 as well as any form of Dlx2 that results from processing in the cell. The term also encompasses naturally occurring variants of Dlx2, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human Dlx2 is MTGVFDSLVADMHSTQIAASSTYHQHQQPPSGGGAGPGGNSSSSSSLHKPQESPTLPVSTATDSSYYTNQQHPAGGGGGGGSPYAHMGSYQYQASGLNNVPYSAKSSYDLGYTAAYTSYAPYGTSSSPANNEPEKEDLEPEIRIVNGKPKKVRKPRTIYSSFQLAALQRRFQKTQYLALPERAELAASLGLTQTQVKIWFQNRRSKFKKMWKSGEIPSEQHPGASASPPCASPPVSAPASWDFGVPQRMAGGGGPGSGGSGAGSSGSSPSSAASAFLGNYPWYHQTSGSASHLQATAPLLHPTQTPQPHHHHHHHGGGGAPVSAGTIF (SEQ ID NO: 3; GenBank Accession NP_004396.1) . Additional examples of orthologous Dlx2 genes and encoded Dlx2 proteins from non-human organisms can be found, for example, on the GenBank website: www. ncbi. nlm. nih. gov/gene/1746/ortholog/? scope=7776&term=DLX2.

As used herein, the term “Ils1 polypeptide” refers to Ils1 or a functional derivative of Isl1. The term “insulin gene enhancer protein” or “Isl1” as used herein, refers to any native Isl1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated. The term encompasses unprocessed Isl1 as well as any form of Isl1 that results from processing in the cell. The term also encompasses naturally occurring variants of Isl1, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human Isl1 is MGDMGDPPKKKRLISLCVGCGNQIHDQYILRVSPDLEWHAACLKCAECNQYLDESCTCFVRDGKTYCKRDYIRLYGIKCAKCSIGFSKNDFVMRARSKVYHIECFRCVACSRQLIPGDEFALREDGLFCRADHDVVERASLGAGDPLSPLHPARPLQMAAEPISARQPALRPHVHKQPEKTTRVRTVLNEKQLHTLRTCYAANPRPDALMKEQLVEMTGLSPRVIRVWFQNKRCKDKKRSIMMKQLQQQQPNDKTNIQGMTGTPMVAASPERHDGGLQANPVEVQSYQPPWKVLSDFALQSDIDQPAFQQLVNFSEGGPGSNSTGSEVASMSSQLPDTPNSMVASPIEA (SEQ ID NO: 4; GenBank Accession: NP_002193.2) . Additional examples of orthologous Isl1 genes and encoded Isl1 proteins from non-human organisms can be found, for example, on the GenBank website: www. ncbi. nlm. nih. gov/gene/3670/ortholog/? scope=89593&term=ISL1.

As used herein, the term “Ascl1 polypeptide” refers to Ascl1 or a functional derivative of Ascl1. The term “achaete-scute family bHLH transcription factor 1” or “Ascl1” as used herein, refers to any native Ascl1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated. The term encompasses unprocessed Ascl1 as well as any form of Ascl1 that results from processing in the cell. The term also encompasses naturally occurring variants of Ascl1, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human Ascl1 is MESSAKMESGGAGQQPQPQPQQPFLPPAACFFATAAAAAAAAAAAAAQSAQQQQQQQQQQQQAPQLRPAADGQPSGGGHKSAPKQVKRQRSSSPELMRCKRRLNFSGFGYSLPQQQPAAVARRNERERNRVKLVNLGFATLREHVPNGAANKKMSKVETLRSAVEYIRALQQLLDEHDAVSAAFQAGVLSPTISPNYSNDLNSMAGSPVSSYSSDEGSYDPLSPEEQELLDFTNWF (SEQ ID NO: 5; GenBank Accession: NP_004307.2) . Additional examples of orthologous Ascl1 genes and encoded Ascl1 proteins from non-human organisms can be found, for example, on the GenBank website: www. ncbi. nlm. nih. gov/gene/429/ortholog/? scope=7776&term=ASCL1.

As used herein, the term “Lhx3 polypeptide” refers to Lhx3 or a functional derivative of Lhx3. The term “LIM homeobox 3” or “lhx3” as used herein, refers to any native lhx3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated. The term encompasses unprocessed lhx3 as well as any form of lhx3 that results from processing in the cell. The term also encompasses naturally occurring variants of lhx3, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human lhx3 isoform a is MLLETGLERDRARPGAAAVCTLGGTREIPLCAGCDQHILDRFILKALDRHWHSKCLKCSDCHTPLAERCFSRGESVYCKDDFFKRFGTKCAACQLGIPPTQVVRRAQDFVYHLHCFACVVCKRQLATGDEFYLMEDSRLVCKADYETAKQREAEATAKRPRTTITAKQLETLKSAYNTSPKPARHVREQLSSETGLDMRVVQVWFQNRRAKEKRLKKDAGRQRWGQYFRNMKRSRGGSKSDKDSVQEGQDSDAEVSFPDEPSLAEMGPANGLYGSLGEPTQALGRPSGALGNFSLEHGGLAGPEQYRELRPGSPYGVPPSPAAPQSLPGPQPLLSSLVYPDTSLGLVPSGAPGGPPPMRVLAGNGPSSDLSTGSSGGYPDFPASPASWLDEVDHAQF (SEQ ID NO: 6; GenBank Accession: NP_835258.1) . Additional examples of orthologous lhx3 genes and encoded lhx3 proteins from non-human organisms can be found, for example, on the GenBank website: www. ncbi. nlm. nih. gov/gene/8022/ortholog/? scope=89593&term=LHX3.

As used herein, the term “Ngn2 polypeptide” refers to Ngn2 or a functional derivative of Ngn2. The term “neurogenin 2” or “Ngn2” as used herein, refers to any native Ngn2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated. The term encompasses unprocessed Ngn2 as well as any form of Ngn2 that results from processing in the cell. The term also encompasses naturally occurring variants of Ngn2, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human Ngn2 is MFVKSETLELKEEEDVLVLLGSASPALAALTPLSSSADEEEEEEPGASGGARRQRGAEAGQGARGGVAAGAEGCRPARLLGLVHDCKRRPSRARAVSRGAKTAETVQRIKKTRRLKANNRERNRMHNLNAALDALREVLPTFPEDAKLTKIETLRFAHNYIWALTETLRLADHCGGGGGGLPGALFSEAVLLSPGGASAALSSSGDSPSPASTWSCTNSPAPSSSVSSNSTSPYSCTLSPASPAGSDMDYWQPPPPDKHRYAPHLPIARDCI (SEQ ID NO: 7; GenBank Accession: NP_076924.1) .

As used herein, the term “LMX1A polypeptide” refers to LMX1A or a functional derivative of LMX1A. The term “LIM homeobox transcription factor 1 alpha” or “LMX1A” as used herein, refers to any native LMX1A from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated. The term encompasses unprocessed LMX1A as well as any form of LMX1A that results from processing in the cell. The term also encompasses naturally occurring variants of LMX1A, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human LMX1A is MLDGLKMEENFQSAIDTSASFSSLLGRAVSPKSVCEGCQRVILDRFLLRLNDSFWHEQCVQCASCKEPLETTCFYRDKKLYCKYDYEKLFAVKCGGCFEAIAPNEFVMRAQKSVYHLSCFCCCVCERQLQKGDEFVLKEGQLLCKGDYEKERELLSLVSPAASDSGKSDDEESLCKSAHGAGKGTAEEGKDHKRPKRPRTILTTQQRRAFKASFEVSSKPCRKVRETLAAETGLSVRVVQVWFQNQRAKMKKLARRQ QQQQQDQQNTQRLSSAQTNGGGSAGMEGIMNPYTALPTPQQLLAIEQSVYSSDPFRQGLTPPQMPGDHMHPYGAEPLFHDLDSDDTSLSNLGDCFLATSEAGPLQSRVGNPIDHLYSMQNSYFTS (SEQ ID NO: 8; GenBank Accession: NP_796372.1) . Additional examples of orthologous LMX1A genes and encoded LMX1A proteins from non-human organisms can be found, for example, on the GenBank website: www. ncbi. nlm. nih. gov/gene/4009/ortholog/? scope=7776&term=LMX1A.

As used herein, the term “Ctip2 polypeptide” refers to Ctip2 or a functional derivative of Ctip2. The term “Ctip2” as used herein, refers to any native Ctip2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated. The term encompasses unprocessed Ctip2 as well as any form of Ctip2 that results from processing in the cell. The term also encompasses naturally occurring variants of Ctip2, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human Ctip2 is MSRRKQGNPQHLSQRELITPEADHVEAAILEEDEGLEIEEPSGLGLMVGGPDPDLLTCGQCQMNFPLGDILVFIEHKRKQCGGSLGACYDKALDKDSPPPSSRSELRKVSEPVEIGIQVTPDEDDHLLSPTKGICPKQENIAGPCRPAQLPAVAPIAASSHPHSSVITSPLRALGALPPCLPLPCCSARPVSGDGTQGEGQTEAPFGCQCQLSGKDEPSSYICTTCKQPFNSAWFLLQHAQNTHGFRIYLEPGPASSSLTPRLTIPPPLGPEAVAQSPLMNFLGDSNPFNLLRMTGPILRDHPGFGEGRLPGTPPLFSPPPRHHLDPHRLSAEEMGLVAQHPSAFDRVMRLNPMAIDSPAMDFSRRLRELAGNSSTPPPVSPGRGNPMHRLLNPFQPSPKSPFLSTPPLPPMPPGGTPPPQPPAKSKSCEFCGKTFKFQSNLIVHRRSHTGEKPYKCQLCDHACSQASKLKRHMKTHMHKAGSLAGRSDDGLSAASSPEPGTSELAGEGLKAADGDFRHHESDPSLGHEPEEEDEEEEEEEEELLLENESRPESSFSMDSELSRNRENGGGGVPGVPGAGGGAAKALADEKALVLGKVMENVGLGALPQYGELLADKQKRGAFLKRAAGGGDAGDDDDAGGCGDAGAGGAVNGRGGGFAPGTEPFPGLFPRKPAPLPSPGLNSAAKRIKVEKDLELPPAALIPSENVYSQWLVGYAASRHFMKDPFLGFTDARQSPFATSSEHSSENGSLRFSTPPGDLLDGGLSGRSGTASGGSTPHLGGPGPGRPSSKEGRRSDTCEYCGKVFKNCSNLTVHRRSHTGERPYKCELCNYACAQSSKLTRHMKTHGQIGKEVYRCDICQMPFSVYSTLEKHMKKWHGEHLLTNDVKIEQAERS (SEQ ID NO: 9; GenBank Accession: XP_054232599.1) .

As used herein, the term “Math5 polypeptide” refers to Math5 or a functional derivative of Math5. The term “Math5” as used herein, refers to any native Math5 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated. The term encompasses unprocessed Math5 as well as any form of Math5 that results from processing in the cell. The term also encompasses naturally occurring variants of Math5, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human Math5 is MKSCKPSGPPAGARVAPPCAGGTECAGTCAGAGRLESAARRRLAANARERRRMQGLNTAFDRL RRVVPQWGQDKKLSKYETLQMALSYIMALTRILAEAERFGSERDWVGLHCEHFGRDHYLPFPGAKLPGESELYSQRLFGFQPEPFQMAT (SEQ ID NO: 10; GenBank Accession: NP_660161.1) .

As used herein, the term “Brn3a polypeptide” refers to Brn3a or a functional derivative of Brn3a. The term “Brn3a” as used herein, refers to any native Brn3a from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated. The term encompasses unprocessed Brn3a as well as any form of Brn3a that results from processing in the cell. The term also encompasses naturally occurring variants of Brn3a, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human Brn3a is MMSMNSKQPHFAMHPTLPEHKYPSLHSSSEAIRRACLPTPPLQSNLFASLDETLLARAEALAAVDIAVSQGKSHPFKPDATYHTMNSVPCTSTSTVPLAHHHHHHHHHQALEPGDLLDHISSPSLALMAGAGGAGAAAGGGGAHDGPGGGGGPGGGGGPGGGPGGGGGGGPGGGGGGPGGGLLGGSAHPHPHMHSLGHLSHPAAAAAMNMPSGLPHPGLVAAAAHHGAAAAAAAAAAGQVAAASAAAAVVGAAGLASICDSDTDPRELEAFAERFKQRRIKLGVTQADVGSALANLKIPGVGSLSQSTICRFESLTLSHNNMIALKPILQAWLEEAEGAQREKMNKPELFNGGEKKRKRTSIAAPEKRSLEAYFAVQPRPSSEKIAAIAEKLDLKKNVVRVWFCNQRQKQKRMKFSATY (SEQ ID NO: 11; GenBank Accession: NP_006228.3) .

A “modification” of an amino acid residue/position refers to a change of a primary amino acid sequence as compared to a starting amino acid sequence, wherein the change results from a sequence alteration involving said amino acid residue/position. For example, typical modifications include substitution of the residue with another amino acid (e.g., a conservative or substantial substitution) , insertion of one or more (e.g., generally fewer than 5, 4, or 3) amino acids adjacent to said residue/position, and/or deletion of said residue/position.

Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Alternatively, naturally occurring residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Generally, conservative substitutions in the sequences of the peptides or polypeptides the disclosure do not abrogate the biological activity of interest of the peptide or polypeptide. Amino acid substitutions may be introduced into a polypeptide of interest and the products screened for a desired activity of interest, e.g., retained/improved ability of a NeuroD1 variant in producing one or more neuronal phenotypes in a glia cell, and methods for measuring such desired activity are well-known in the art.

In contrast, substantial modifications in the biological properties of a polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

In the context of a peptide or polypeptide, the term “derivative” as used herein refers to a peptide or polypeptide that comprises an amino acid sequence of the peptide or polypeptide, or a fragment of a peptide or polypeptide, which has been altered by the introduction of amino acid residue substitutions, deletions, or additions. The term “derivative” as used herein also refers to a peptide or polypeptide, or a fragment of a peptide or polypeptide, which has been chemically modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, a peptide or polypeptide or a fragment of the peptide or polypeptide may be chemically modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, chemical cleavage, formulation, metabolic synthesis of tunicamycin, linkage to a cellular ligand or other protein, etc. The derivatives are modified in a manner that is different from naturally occurring or starting peptide or polypeptides, either in the type or location of the molecules attached. Derivatives further include deletion of one or more chemical groups which are naturally present on the peptide or polypeptide. Further, a derivative of a peptide or polypeptide or a fragment of a peptide or polypeptide may contain one or more non-classical amino acids. In specific embodiments, a derivative is a functional derivative of the native or unmodified peptide or polypeptide (e.g., a wild-type protein) from which it was derived. For example, a derivative of human NeuroD1 contains one or more modifications in its amino acid sequence with respect to the sequence shown in SEQ ID NO: 1.

The term “functional derivative” refers to a derivative that retains one or more functions or activities of the naturally occurring or starting peptide or polypeptide (e.g. a wild-type protein) from which it is derived. For example, in some embodiments, a functional derivative of a reprograming protein factor as described herein (e.g., NeuroD1) may retain the activity of producing a neuronal phenotype in a glial cell after being expressed in a sufficient amount by the glial cell. In some embodiments, a functional derivative of a reprogramming protein factor may retain the activity of the reprogramming protein factor in reprogramming the glial cell to trans-differentiate into a functional neuron after being expressed in a sufficient amount by the glial cell. In some embodiments, a functional derivative of a peptide or polypeptide described herein shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the starting (e.g., wild-type) peptide or polypeptide.

A derivative of polypeptide can be prepared using methods well-known in the art, e.g., by modifying the corresponding nucleic acid molecules encoding the derivative. For example, derivatives may be a substitution, deletion, or insertion of one or more codons encoding the polypeptide that results in a change in the amino acid sequence as compared with the wild-type sequence of the polypeptide. The derivatives can be made using methods well-known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter, 1986, Biochem J. 237: 1-7; and Zoller et al., 1982, Nucl. Acids Res. 10: 6487-500) , cassette mutagenesis (see, e.g., Wells et al., 1985, Gene 34: 315-23) , or other known techniques can be performed on the cloned DNA to produce the derivatives DNA.

Those skilled in the art can determine the site (s) in an amino acid sequence of a given protein, where a modification (s) can be made in order to produce functional derivatives. In some embodiments, a functional derivative of a polypeptide comprises one or more modifications to one or more predicted non-essential amino acid residues in its sequence. In some embodiments, modifications made to non-essential amino acid residues can be a conservative substation as described herein. In some embodiments, modifications made to non-essential amino acid residues can be a substantial substation described herein. In some embodiments, modifications made to non-essential amino acid residues can be a deletion of the non-essential amino acid residue. In alternative embodiments, one or more modifications can be made to one or more predicted essential amino acid residues in its sequence. In particularly embodiments, the modifications made to essential amino acid residues in a protein sequence can be a conservative substitution as described herein. Methods well-known in the art can be used to analyze a protein (e.g., NeuroD1) sequence to identify essential and non-essential amino acid residues of the protein. For example, in some embodiments, an amino acid residue of a protein that is not conserved among orthologous gene products is predicted to be a non-essential amino acid residue, while another amino acid residue that is conserved among orthologous gene products is predicted to be an essential amino acid residue. For example, an alignment of twelve NeuroD1 orthologs is shown in Figure 7, and the conserved residues and non-conserved residues are marked with different shades, respectively.

In some embodiments, after making one or more modifications to the sequence of a polypeptide (e.g., by making insertions, deletions, or substitutions of amino acids in the original amino acid sequence either systematically, randomly, or at selected sites) , functional derivatives of the polypeptide can be identified by testing the resulting derivatives for activity exhibited by the original sequence. For example, to identify functional derivative of a reprograming protein factor (e.g., NeuroD1) as described herein, nucleic acid molecules encoding the derivative polypeptides can be delivered into a population of starting glial cells under a suitable condition to be expressed at a sufficient level, and assays can be conducted to detect and/or measure one or more neuronal phenotypes in the population of cells and compared the level at which the neuronal phenotype of interest is demonstrated by the population of cells to a control group of glial cells that express the original, unmodified (e.g., wild-type) reprogramming protein factor, and those derivatives that induce the neuronal phenotype in the testing cell population at a comparable level to that of the control population can be selected as functional derivatives. Alternatively, the comparison can be made to a control group of glial cells that do not express the reprogramming protein factor (e.g. transfected with a blank vector) , and those derivative that induce the neuronal phenotype in the testing cell population at a greater level than that of the control population can be selected as functional derivatives.

The term “sequence identity” refers to a relationship between the sequences of two or more biological molecules (e.g., a pair of polynucleotides or multiple polypeptides) , as determined by aligning and comparing the respective sequences. “Percent (%) amino acid sequence identity” with respect to a reference amino acid sequence (e.g., a reference polypeptide) is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference amino acid sequence, after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNAStar, Inc. ) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sept-16-1998) and the following parameters: Match: 1; mismatch: -2; gap open: 5; gap extension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.

The term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding a peptide or protein as described herein, in order to introduce a nucleic acid sequence into a host cell, or serve as a transcription template to carry out in vitro transcription reaction in a cell-free system to produce mRNA. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate transcription or translation control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Transcription or translation control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are to be co-transcribed or co-translated (e.g., nucleic acid molecules encoding two or more different peptides or proteins) , both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector transcription and/or translation, the encoding nucleic acids can be operationally linked to one common transcription or translation control sequence or linked to different transcription or translation control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product (e.g., a mRNA transcript of the nucleic acid as described herein) , and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

The term “administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a lipid nanoparticle composition as described herein) into a patient, such as by intracranial, mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art. When a disease, disorder, condition, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease, disorder, condition, or symptoms thereof. When a disease, disorder, condition, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease, disorder, condition, or symptoms thereof.

“Chronic” administration refers to administration of the agent (s) in a continuous mode (e.g., for a period of time such as days, weeks, months, or years) as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

The term “targeted delivery” or the verb form “target” as used herein refers to the process that promotes the arrival of a delivered agent (such as a therapeutic payload molecule in a lipid nanoparticle composition as described herein) at a specific organ, tissue, cell and/or intracellular compartment (referred to as the targeted location) more than any other organ, tissue, cell or intracellular compartment (referred to as the non-target location) . Targeted delivery can be detected using methods known in the art, for example, by comparing the concentration of the delivered agent in a targeted cell population with the concentration of the delivered agent at a non-target cell population after systemic administration. In certain embodiments, targeted delivery results in at least 2 fold higher concentration at a targeted location as compared to a non-target location.

An “effective amount” is generally an amount sufficient to produce a desirable outcome, such as, producing one or more neuronal phenotypes in a population of cells, or in the context of disease management, to reduce the severity and/or frequency of symptoms, eliminate the symptoms and/or underlying cause, prevent the occurrence of symptoms and/or their underlying cause, and/or improve or remediate the damage that results from or is associated with a disease, disorder, or condition, including, for example, infection and neoplasia.

The terms “subject” and “patient” may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc. ) or a primate (e.g., monkey and human) . In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) having an infectious disease or neoplastic disease. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing an infectious disease or neoplastic disease.

The term “phenotype” refers to well-known detectable characteristics of the cells referred to herein. The neuronal phenotype can be, but is not limited to, one or more of: neuronal morphology, expression of one or more neuronal markers, electrophysiological characteristics of neurons, synapse formation and release of neurotransmitter. For example, neuronal phenotype encompasses but is not limited to: characteristic morphological aspects of a neuron such as presence of dendrites, an axon and dendritic spines; characteristic neuronal protein expression and distribution, such as presence of synaptic proteins in synaptic puncta, presence of MAP2 in dendrites; and characteristic electrophysiological signs such as spontaneous and evoked synaptic events. Phenotypes that distinguish a neuron from a non- neuron cell (e.g., a glial cell) as well as method for detecting and measuring such phenotypes are known to those of ordinary skill in the art.

The term “detectable probe” refers to a composition that provides a detectable signal. The term includes, without limitation, any fluorophore, chromophore, radiolabel, enzyme, antibody or antibody fragment, and the like, that provide a detectable signal via its activity.

The term “detectable agent” refers to a substance that can be used to ascertain the existence or presence of a desired molecule, such as an antigen encoded by an mRNA molecule as described herein, in a sample or subject. A detectable agent can be a substance that is capable of being visualized or a substance that is otherwise able to be determined and/or measured (e.g., by quantitation) .

As used herein and unless otherwise specified, the term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many nonpolar organic solvents. While lipids generally have poor solubility in water, there are certain categories of lipids (e.g., lipids modified by polar groups, e.g., DMG-PEG2000) that have limited aqueous solubility and can dissolve in water under certain conditions. Known types of lipids include biological molecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids can be divided into at least three classes: (1) “simple lipids, ” which include fats and oils as well as waxes; (2) “compound lipids, ” which include phospholipids and glycolipids (e.g., DMPE-PEG2000) ; and (3) “derived lipids” such as steroids. Further, as used herein, lipids also encompass lipidoid compounds. The term “lipidoid compound, ” also simply “lipidoid” , refers to a lipid-like compound (e.g. an amphiphilic compound with lipid-like physical properties) .

The term “lipid nanoparticle” or “LNP” refers to a particle having at least one dimension on the order of nanometers (nm) (e.g., 1 to 1,000 nm) , which contains one or more types of lipid molecules. The LNP provided herein can further contain at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules) . In some embodiments, the LNP comprises a non-lipid payload molecule either partially or completely encapsulated inside a lipid shell. Particularly, in some embodiments, wherein the payload is a negatively charged molecule (e.g., mRNA encoding a protein) , and the lipid components of the LNP comprise at least one cationic lipid. Without being bound by the theory, it is contemplated that the cationic lipids can interact with the negatively charged payload molecules and facilitates incorporation and/or encapsulation of the payload into the LNP during LNP formation. Other lipids that can form part of a LNP as provided herein include but are not limited to neutral lipids and charged lipids, such as steroids, polymer conjugated lipids, and various zwitterionic lipids. In certain embodiments, a LNP according to the present disclosure comprises one or more lipids of Formula (I) to (IV) (and sub-formulas thereof) as described herein.

The term “cationic lipid” refers to a lipid that is either positively charged at any pH value or hydrogen ion activity of its environment, or capable of being positively charged in response to the pH value or hydrogen ion activity of its environment (e.g., the environment of its intended use) . Thus, the term “cationic” encompasses both “permanently cationic” and “cationisable. ” In certain embodiments, the positive charge in a cationic lipid results from the presence of a quaternary nitrogen atom. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge in the environment of its intended use (e.g., at physiological pH) .

The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid (PEG-lipid) , in which the polymer portion comprises a polyethylene glycol.

The term “neutral lipid” encompasses any lipid molecules existing in uncharged forms or neutral zwitterionic forms at a selected pH value or within a selected pH range. In some embodiments, the selected useful pH value or range corresponds to the pH condition in an environment of the intended uses of the lipids, such as the physiological pH. As non-limiting examples, neutral lipids that can be used in connection with the present disclosure include, but are not limited to, phosphotidylcholines such as 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) , 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) , 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) , 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) , 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) , phophatidylethanolamines such as 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) , 2- ( (2, 3-bis (oleoyloxy) propyl) dimethylammonio) ethyl hydrogen phosphate (DOCP) , sphingomyelins (SM) , ceramides, steroids such as sterols and their derivatives. Neutral lipids as provided herein may be synthetic or derived (isolated or modified) from a natural source or compound.

The term “charged lipid” encompasses any lipid molecules that exist in either positively charged or negatively charged forms at a selected pH or within a selected pH range. In some embodiments, the selected pH value or range corresponds to the pH condition in an environment of the intended uses of the lipids, such as the physiological pH. As non-limiting examples, neutral lipids that can be used in connection with the present disclosure include, but are not limited to, phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, sterol hemisuccinates, dialkyl trimethylarnmonium-propanes, (e.g., DOTAP, DOTMA) , dialkyl dimethylaminopropanes, ethyl phosphocholines, dimethylaminoethane carbamoyl sterols (e.g., DC-Chol) , 1, 2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt (DOPS-Na) , 1, 2-dioleoyl-sn-glycero-3-phospho- (1'-rac-glycerol) sodium salt (DOPG-Na) , and 1, 2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPA-Na) . Charged lipids as provided herein may be synthetic or derived (isolated or modified) from a natural source or compound.

As used herein, and unless otherwise specified, the term “alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated. In one embodiment, the alkyl group has, for example, from one to twenty-four carbon atoms (C₁-C₂₄ alkyl) , four to twenty carbon atoms (C₄-C₂₀ alkyl) , six to sixteen carbon atoms (C₆-C₁₆ alkyl) , six to nine carbon atoms (C₆-C₉ alkyl) , one to fifteen carbon atoms (C₁-C₁₅ alkyl) , one to twelve carbon atoms (C₁-C₁₂ alkyl) , one to eight carbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆ alkyl) and which is attached to the rest of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl) , n-butyl, n-pentyl, 1, 1-dimethylethyl (t-butyl) , 3-methylhexyl, 2-methylhexyl, and the like. Unless otherwise specified, an alkyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “alkenyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which contains one or more carbon-carbon double bonds. The term “alkenyl” also embraces radicals having “cis” and “trans” configurations, or alternatively, “E” and “Z” configurations, as appreciated by those of ordinary skill in the art. In one embodiment, the alkenyl group has, , for example, from two to twenty-four carbon atoms (C₂-C₂₄ alkenyl) , four to twenty carbon atoms (C₄-C₂₀ alkenyl) , six to sixteen carbon atoms (C₆-C₁₆ alkenyl) , six to nine carbon atoms (C₆-C₉ alkenyl) , two to fifteen carbon atoms (C₂-C₁₅ alkenyl) , two to twelve carbon atoms (C₂-C₁₂ alkenyl) , two to eight carbon atoms (C₂-C₈ alkenyl) or two to six carbon atoms (C₂-C₆ alkenyl) and which is attached to the rest of the molecule by a single bond. Examples of alkenyl groups include, but are not limited to, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1, 4-dienyl, and the like. Unless otherwise specified, an alkenyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “alkynyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which contains one or more carbon-carbon triple bonds. In one embodiment, the alkynyl group has, for example, from two to twenty-four carbon atoms (C₂-C₂₄ alkynyl) , four to twenty carbon atoms (C₄-C₂₀ alkynyl) , six to sixteen carbon atoms (C₆-C₁₆ alkynyl) , six to nine carbon atoms (C₆-C₉ alkynyl) , two to fifteen carbon atoms (C₂-C₁₅ alkynyl) , two to twelve carbon atoms (C₂-C₁₂ alkynyl) , two to eight carbon atoms (C₂-C₈ alkynyl) or two to six carbon atoms (C₂-C₆ alkynyl) and which is attached to the rest of the molecule by a single bond. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like. Unless otherwise specified, an alkynyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which is saturated. In one embodiment, the alkylene has, for example, from one to twenty-four carbon atoms (C₁-C₂₄ alkylene) , one to fifteen carbon atoms (C₁- C₁₅ alkylene) , one to twelve carbon atoms (C₁-C₁₂ alkylene) , one to eight carbon atoms (C₁-C₈ alkylene) , one to six carbon atoms (C₁-C₆ alkylene) , two to four carbon atoms (C₂-C₄ alkylene) , one to two carbon atoms (C₁-C₂ alkylene) . Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, n-butylene, and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless otherwise specified, an alkylene chain is optionally substituted.

As used herein, and unless otherwise specified, the term “alkenylene” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which contains one or more carbon-carbon double bonds. In one embodiment, the alkenylene has, for example, from two to twenty-four carbon atoms (C₂-C₂₄ alkenylene) , two to fifteen carbon atoms (C₂-C₁₅ alkenylene) , two to twelve carbon atoms (C₂-C₁₂ alkenylene) , two to eight carbon atoms (C₂-C₈ alkenylene) , two to six carbon atoms (C₂-C₆ alkenylene) or two to four carbon atoms (C₂-C₄ alkenylene) . Examples of alkenylene include, but are not limited to, ethenylene, propenylene, n-butenylene, and the like. The alkenylene is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkenylene to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless otherwise specified, an alkenylene is optionally substituted.

As used herein, and unless otherwise specified, the term “cycloalkyl” refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, and which is saturated. Cycloalkyl group may include fused or bridged ring systems. In one embodiment, the cycloalkyl has, for example, from 3 to 15 ring carbon atoms (C₃-C₁₅ cycloalkyl) , from 3 to 10 ring carbon atoms (C₃-C₁₀ cycloalkyl) , or from 3 to 8 ring carbon atoms (C₃-C₈ cycloalkyl) . The cycloalkyl is attached to the rest of the molecule by a single bond. Examples of monocyclic cycloalkyl radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyl radicals include, but are not limited to, adamantyl, norbornyl, decalinyl, 7, 7-dimethyl-bicyclo [2.2.1] heptanyl, and the like. Unless otherwise specified, a cycloalkyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “cycloalkylene” is a divalent cycloalkyl group. Unless otherwise specified, a cycloalkylene group isoptionally substituted.

As used herein, and unless otherwise specified, the term “cycloalkenyl” refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, and which includes one or more carbon-carbon double bonds. Cycloalkenyl may include fused or bridged ring systems. In one embodiment, the cycloalkenyl has, for example, from 3 to 15 ring carbon atoms (C₃- C₁₅ cycloalkenyl) , from 3 to 10 ring carbon atoms (C₃-C₁₀ cycloalkenyl) , or from 3 to 8 ring carbon atoms (C₃-C₈ cycloalkenyl) . The cycloalkenyl is attached to the rest of the molecule by a single bond. Examples of monocyclic cycloalkenyl radicals include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like. Unless otherwise specified, a cycloalkenyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “cycloalkenylene” is a divalent cycloalkenyl group. Unless otherwise specified, a cycloalkenylene group is optionally substituted.

As used herein, and unless otherwise specified, the term “heterocyclyl” refers to a non-aromatic radical monocyclic or polycyclic moiety that contains one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from nitrogen, oxygen, phosphorous, and sulfur. The heterocyclyl may be attached to the main structure at any heteroatom or carbon atom. A heterocyclyl group can be a monocyclic, bicyclic, tricyclic, tetracyclic, or other polycyclic ring system, wherein the polycyclic ring systems can be a fused, bridged or spiro ring system. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or more rings. A heterocyclyl group can be saturated or partially unsaturated. Saturated heterocycloalkyl groups can be termed “heterocycloalkyl” . Partially unsaturated heterocycloalkyl groups can be termed “heterocycloalkenyl” if the heterocyclyl contains at least one double bond, or “heterocycloalkynyl” if the heterocyclyl contains at least one triple bond. In one embodiment, the heterocyclyl has, for example, 3 to 18 ring atoms (3-to 18-membered heterocyclyl) , 4 to 18 ring atoms (4-to 18-membered heterocyclyl) , 5 to 18 ring atoms (3-to 18-membered heterocyclyl) , 4 to 8 ring atoms (4-to 8-membered heterocyclyl) , or 5 to 8 ring atoms (5-to 8-membered heterocyclyl) . Whenever it appears herein, a numerical range such as “3 to 18” refers to each integer in the given range; e.g., “3 to 18 ring atoms” means that the heterocyclyl group can consist of 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, etc., up to and including 18 ring atoms. Examples of heterocyclyl groups include, but are not limited to, imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl. Unless otherwise specified, a heterocyclyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “heterocyclylene” is a divalent heterocyclyl group. Unless otherwise specified, a heterocyclylene group is optionally substituted

As used herein, and unless otherwise specified, the term “aryl” refers to a monocyclic aromatic group and/or multicyclic monovalent aromatic group that contain at least one aromatic hydrocarbon ring. In certain embodiments, the aryl has from 6 to 18 ring carbon atoms (C₆-C₁₈ aryl) , from 6 to 14 ring carbon atoms (C₆-C₁₄ aryl) , or from 6 to 10 ring carbon atoms (C₆-C₁₀ aryl) . Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl. The term “aryl” also refers to bicyclic, tricyclic, or other multicyclic hydrocarbon rings, where at least one of the rings is aromatic and the others of which may be saturated, partially unsaturated, or aromatic, for example, dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetralinyl) . Unless otherwise specified, an aryl group is optionally substituted.

As used herein, and unless otherwise specified, the term “arylene” is a divalent aryl group. Unless otherwise specified, an arylene group is optionally substituted.

As used herein, and unless otherwise specified, the term “heteroaryl” refers to a monocyclic aromatic group and/or multicyclic aromatic group that contains at least one aromatic ring, wherein at least one aromatic ring contains one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from O, S, and N. The heteroaryl may be attached to the main structure at any heteroatom or carbon atom. In certain embodiments, the heteroaryl has from 5 to 20, from 5 to 15, or from 5 to 10 ring atoms. The term “heteroaryl” also refers to bicyclic, tricyclic, or other multicyclic rings, where at least one of the rings is aromatic and the others of which may be saturated, partially unsaturated, or aromatic, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, S, and N. Examples of monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl. Examples of bicyclic heteroaryl groups include, but are not limited to, indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, purinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl, and tetrahydroquinolinyl. Examples of tricyclic heteroaryl groups include, but are not limited to, carbazolyl, benzindolyl, phenanthrollinyl, acridinyl, phenanthridinyl, and xanthenyl. Unless otherwise specified, a heteroaryl group is optionally substituted.

As used herein, and unless otherwise specified, the term “heteroarylene” is a divalent heteroaryl group. Unless otherwise specified, a heteroarylene group is optionally substituted.

When the groups described herein are said to be “substituted, ” they may be substituted with any appropriate substituent or substituents. Illustrative examples of substituents include, but are not limited to, those found in the exemplary compounds and embodiments provided herein, as well as: a halogen atom such as F, CI, Br, or I; cyano; oxo (=O) ; hydroxyl (-OH) ; alkyl; alkenyl; alkynyl; cycloalkyl; aryl; - (C=O) OR’; -O (C=O) R’; -C (=O) R’; -OR’; -S (O) _xR’; -S-SR’; -C (=O) SR’; -SC (=O) R’; -NR’R’; -NR’C (=O) R’; -C (=O) NR’R’; -NR’C (=O) NR’R’; -OC (=O) NR’R’; -NR’C (=O) OR’; -NR’S (O) _xNR’R’; -NR’S (O) _xR’; and -S (O) _xNR’R’, wherein: R’ is, at each occurrence, independently H, C₁- C₁₅ alkyl or cycloalkyl, and x is 0, 1 or 2. In some embodiments the substituent is a C₁-C₁₂ alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group (-OR’) . In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amino group (-NR’R’) .

As used herein, and unless otherwise specified, the term “optional” or “optionally” (e.g., optionally substituted) means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally wherein” means that the features following the wherein may or may not be present and that the description includes either situation when such features are present or absent.

As used herein, and unless otherwise specified, the term “prodrug” of a biologically active compound refers to a compound that may be converted under physiological conditions or by solvolysis to the biologically active compound. In one embodiment, the term “prodrug” refers to a metabolic precursor of the biologically active compound that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo to the biologically active compound. Prodrugs are typically rapidly transformed in vivo to yield the parent biologically active compound, for example, by hydrolysis in blood. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgard, H., Design of Prodrugs (1985) , pp. 7-9, 21-24 (Elsevier, Amsterdam) ) . A discussion of prodrugs is provided in Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

In one embodiment, the term “prodrug” is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a mammalian subject. Prodrugs of a compound may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Prodrugs include compounds wherein a hydroxyl, amino or mercapto group is bonded to any group that, when the prodrug of the compound is administered to a mammalian subject, cleaves to form a free hydroxyl, free amino or free mercapto group, respectively.

Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol or amide derivatives of amine functional groups in the compounds provided herein.

As used herein, and unless otherwise specified, the term “pharmaceutically acceptable salt” includes both acid and base addition salts.

Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2, 2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1, 5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

Examples of pharmaceutically acceptable base addition salt include, but are not limited to, salts prepared from addition of an inorganic base or an organic base to a free acid compound. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. In one embodiment, the inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. In one embodiment, the organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

A compound provided herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R) -or (S) -or, as (D) -or (L) -for amino acids. Unless otherwise specified, a compound provided herein is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-) , (R) -and (S) -, or (D) -and (L) -isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC) . When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

As used herein, and unless otherwise specified, the term “isomer” refers to different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space. “Atropisomers” are stereoisomers from hindered rotation about single bonds. “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. A mixture of a pair of enantiomers in any proportion can be known as a “racemic” mixture. “Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other.

“Stereoisomers” can also include E and Z isomers, or a mixture thereof, and cis and trans isomers or a mixture thereof. In certain embodiments, a compound described herein is isolated as either the E or Z isomer. In other embodiments, a compound described herein is a mixture of the E and Z isomers.

“Tautomers” refers to isomeric forms of a compound that are in equilibrium with each other. The concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution.

It should also be noted a compound described herein can contain unnatural proportions of atomic isotopes at one or more of the atoms. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (³H) , iodine-125 (¹²⁵I) , sulfur-35 (³⁵S) , or carbon-14 (¹⁴C) , or may be isotopically enriched, such as with deuterium (²H) , carbon-13 (¹³C) , or nitrogen-15 (¹⁵N) . As used herein, an “isotopolog” is an isotopically enriched compound. The term “isotopically enriched” refers to an atom having an isotopic composition other than the natural isotopic composition of that atom. “Isotopically enriched” may also refer to a compound containing at least one atom having an isotopic composition other than the natural isotopic composition of that atom. The term “isotopic composition” refers to the amount of each isotope present for a given atom. Radiolabeled and isotopically enriched compounds are useful as therapeutic agents, e.g., cancer therapeutic agents, research reagents, e.g., binding assay reagents, and diagnostic agents, e.g., in vivo imaging agents. All isotopic variations of a compound described herein, whether radioactive or not, are intended to be encompassed within the scope of the embodiments provided herein. In some embodiments, there are provided isotopologs of a compound described herein, for example, the isotopologs are deuterium, carbon-13, and/or nitrogen-15 enriched. As used herein, “deuterated” , means a compound wherein at least one hydrogen (H) has been replaced by deuterium (indicated by D or ²H) , that is, the compound is enriched in deuterium in at least one position.

It should be noted that if there is a discrepancy between a depicted structure and a name for that structure, the depicted structure is to be accorded more weight.

The term “composition” is intended to encompass a product containing the specified ingredients (e.g., a mRNA or circular RNA molecule provided herein) in, optionally, the specified amounts.

“Substantially all” refers to at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

As used herein, and unless otherwise indicated, the term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.05%, or less of a given value or range. As used herein, when “about” is used in connection with a numerical range, the term “about” is meant to apply to both ends of such modified range (e.g., “about 5 to 10” means “about 5 to about 10” ) .

The singular terms “a, ” “an, ” and “the” as used herein include the plural reference unless the context clearly indicates otherwise.

All publications, patent applications, accession numbers, and other references cited in this specification are herein incorporated by reference in their entirety as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the descriptions in the Experimental section and examples are intended to illustrate but not limit the scope of invention described in the claims.

5.3 Functional Nucleic Acids

In one aspect, provided herein are functional nucleic acid molecules for reprogramming a glial cell into a functional neuron. In some embodiments, the functional nucleic acid encodes a peptide or polypeptide, which upon contacting with the glial cell, is expressed by the glial cell to produce the encoded peptide or polypeptide.

In one aspect of the present disclosure, the functional nucleic acid molecules provided herein are, or are configured to produce, a linear nucleic acid molecule, including a single-stranded linear RNA molecule. Accordingly, provided herein are linear RNAs (e.g., linear mRNAs) , and vectors (e.g., DNA vectors) that can be transcribed into the linear RNAs. The linear nucleic acid molecules described in this section can comprise any expression sequence or coding sequences as described in this Section 5.3.1 (Coding Region) . A linear nucleic acid sequence has a 5’ end and a 3’ end. Accordingly, in some embodiments, the functional nucleic acid molecules are DNA molecules. In other embodiments, the functional nucleic acid molecules are RNA molecules. In particular embodiments, the functional nucleic acid molecules are linear RNA molecules. In particular embodiments, the functional nucleic acid molecules are linear mRNA molecules.

According to another aspect of the present disclosure, the functional nucleic acid molecules provided herein are, or are configured to produce, a circular nucleic acid molecule, and particularly a single-stranded circular RNA molecule. Accordingly, provided herein are circular RNAs, precursor RNAs that can circularize into the circular RNAs, and vectors (e.g., DNA vectors) that can be transcribed into the precursor RNAs or the circular RNAs. The circular nucleic acid molecules described herein can comprise any expression sequence or coding sequences as described in Section 5.3.1 (Coding Region) . A circular nucleic acid sequence does not have 5’ end or a 3’ end. Accordingly, in some embodiments, the functional nucleic acid molecules are DNA molecules. In other embodiments, the functional nucleic acid molecules are RNA molecules. In particular embodiments, the functional nucleic acid molecules are linear RNA molecules configured for forming a circular RNA molecule under a suitable condition, and such linear RNA molecules are sometimes referred to as a precursor RNA molecule in this application.

In some embodiments, the functional nucleic acid of the present disclosure comprises an expression sequence encoding at least one protein factor, which upon expression by a glial cell, is capable of reprogramming the glial cell to trans-differentiate into functional neurons. In some embodiments, the functional nucleic acid of the present disclosure comprises an expression sequence encoding at least functional RNA, which upon expression by a glial cell, is capable of reprogramming the glial cell to trans-differentiate into functional neurons. A plurality of protein and RNA factors that can be used in connection with the present disclosure (e.g., to be encoded by the functional nucleic acid provided herein) to reprogram various types of glial cells into various types of functional neurons, and those protein factors are sometimes referred to as a reprogramming protein factor in this application. Various reprogramming factors (including reprogramming protein factors) can be used alone or in combination with one another for this purpose. See, for example, Table 1.

In some embodiment, the functional nucleic acid molecules of the present disclosure comprise at least one coding region encoding a peptide or polypeptide of interest (e.g., an open reading frame (ORF) ) . In some embodiments, the functional nucleic acid molecule further comprises at least one untranslated region (UTR) . In some embodiments, the UTR comprises one or more regulatory elements as described herein. In some embodiments, the UTR contains secondary structure in its nucleic acid sequence, such as one or more stem-loop structures.

5.3.1 Coding Region

In some embodiments, the nucleic acid molecule of the present disclosure comprises at least one coding region. In some embodiments, the coding region is an open reading frame (ORF) that encodes for a single peptide, protein, or functional RNA (e.g. microRNA) . In some embodiments, the coding region comprises at least two ORFs, each encoding a peptide, protein or a functional RNA. In those embodiments where the coding region comprises more than one ORFs, the encoded peptides and/or proteins can be the same as or different from each other. In some embodiments, the multiple ORFs in a coding region are separated by non-coding sequences.

In specific embodiments, the functional nucleic acid of the present disclosure encodes one or more polypeptides that are reprogramming protein factors as described herein. In specific embodiments, the encoded one or more polypeptides comprises a NeuroD1 polypeptide as described herein. Table 2 shows exemplary reprogramming protein factors and encoding nucleic acid sequences.

Table 2 Exemplary NeuroD1 polypeptide and encoding nucleic acid sequences.

In particular embodiments, the nucleic acid of the present disclosure encodes a NeuroD1 polypeptide. In some embodiments, the encoded NeuroD1 polypeptide is a wild-type NeuroD1. In some embodiments, the encoded NeuroD1 is human NeuroD1 having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the encoded NeuroD1 is a NeuroD1 polypeptide having the amino acid sequence of SEQ ID NO: 2, where an extra V encoded by a Kozak fragment is located at the second residue.

In alternative embodiments, the encoded NeuroD1 polypeptide is a functional derivative of NeuroD1. In some embodiments, a functional derivative of NeuroD1 shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) NeuroD1 protein from which it derives.

In some embodiments, a functional derivative of NeuroD1 comprises one or more modifications to one or more predicted non-essential amino acid residues in the NeuroD1 sequence. Methods well-known in the art can be used to analyze a protein (e.g., NeuroD1) sequence to identify essential and non-essential amino acid residues of the protein. For example, in some embodiments, an amino acid residue of a protein that is not conserved among orthologous gene products is predicted to be a non-essential amino acid residue, while another amino acid residue that is conserved among orthologous gene products is predicted to be an essential amino acid residue. An exemplary alignment of NeuroD1 orthologs is shown in Figure 7, and the conserved residues and non-conserved residues are marked with different shades, respectively.

In specific embodiments, a functional derivative of NeuroD1 comprises one or more conservative amino acid substitutions at one or more predicted non-essential amino acid residues of NeuroD1. In specific embodiments, a functional derivative of NeuroD1 comprises one or more conservative amino acid substitutions at one or more predicted essential amino acid residues of NeuroD1.

In some embodiments, a functional derivative of NeuroD1 retains the NeuroD1 function in producing one or more neuronal phenotypes in a glial cell, which neuronal phenotypes include but are not limited to neuronal morphology, expression of one or more neuronal marker, electrophysiologic characteristics of neurons, synapse formation and release of neurotransmitters. Methods disclosed herein (see e.g., Example section) and/or well-known in the art can be used to measure the one or more neuronal phenotypes. In some embodiments, a functional derivative of NeuroD1 retains the NeuroD1 function in reprogramming a glial cell to trans-differentiate into a functional neuron.

In specific embodiments, a functional derivative of NeuroD1 comprises one or more conservative amino acid substitutions at one or more predicted non-essential amino acid residues, and shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to a wild-type NeuroD1 protein. In some embodiments, the wild-type NeuroD1 protein from which the functional derivative is derived is a wild-type human NeuroD1 having SEQ ID NO: 1. In some embodiments, the NeuroD1 protein from which the functional derivative is derived is a NeuroD1 polypeptide having SEQ ID NO: 2.

In specific embodiments, a functional derivative of NeuroD1 comprises one or more conservative amino acid substitutions at one or more predicted non-essential amino acid residues, and shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) NeuroD1 protein from which it derives, and further retains the function in producing one or more neuronal phenotypes in a glial cell when expressed in a sufficient amount by the glial cell. In some embodiments, the wild-type NeuroD1 protein from which the functional derivative is derived is a wild-type human NeuroD1 having SEQ ID NO: 1. In some embodiments, the NeuroD1 protein from which the functional derivative is derived is a NeuroD1 polypeptide having SEQ ID NO: 2.

In specific embodiments, a functional derivative of NeuroD1 comprises one or more conservative amino acid substitutions at one or more predicted non-essential amino acid residues, and shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) NeuroD1 protein from which it derives, and further retains the function in reprogramming a glial cell to trans-differentiate into a functional neuron when expressed in a sufficient amount by the glial cell. In some embodiments, the wild-type NeuroD1 protein from which the functional derivative is derived is a wild-type human NeuroD1 having SEQ ID NO: 1. In some embodiments, the NeuroD1 protein from which the functional derivative is derived is a NeuroD1 polypeptide having SEQ ID NO: 2.

In specific embodiments, a functional derivative of NeuroD1 comprises one or more conservative amino acid substitutions at one or more predicted essential amino acid residues, and shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) NeuroD1 protein from which it derives, and further retains the function in producing one or more neuronal phenotypes in a glial cell when expressed in a sufficient amount by the glial cell. In some embodiments, the wild-type NeuroD1 protein from which the functional derivative is derived is a wild- type human NeuroD1 having SEQ ID NO: 1. In some embodiments, the NeuroD1 protein from which the functional derivative is derived is a NeuroD1 polypeptide having SEQ ID NO: 2.

In specific embodiments, a functional derivative of NeuroD1 comprises one or more conservative amino acid substitutions at one or more predicted essential amino acid residues, and shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) NeuroD1 protein from which it derives, and further retains the function in reprogramming a glial cell to trans-differentiate into a functional neuron when expressed in a sufficient amount by the glial cell. In some embodiments, the wild-type NeuroD1 protein from which the functional derivative is derived is a wild-type human NeuroD1 having SEQ ID NO: 1. In some embodiments, the NeuroD1 protein from which the functional derivative is derived is a NeuroD1 polypeptide having SEQ ID NO: 2.

In some embodiments, the encoded NeuroD1 polypeptide is encoded by (a) a DNA sequence of SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, (b) a codon-optimized variant of (a) , or (c) a transcribed RNA sequence of (a) or (b) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 12. In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 13. In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 14. In some embodiments, the transcribed RNA sequence has the same sequence as the DNA coding sequences except that thymine bases in the DNA sequence are replaced by uracil bases in the RNA sequence.

In some embodiments, the encoded NeuroD1 polypeptide is encoded by (a) a RNA sequence of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, or (b) a codon-optimized variant of (a) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 15. In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 16. In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 17.

In particular embodiments, the nucleic acid of the present disclosure comprises a multi-cistronic (e.g., bi-cistronic or tri-cistronic) expression sequence that encodes a NeuroD1 polypeptide and at least one second polypeptide. In some embodiments, the encoded second polypeptide does not interfere any biological function of the encoded NeuroD1 polypeptide. In various embodiments, the multi-cistronic expression sequence encodes for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 peptides or proteins. Peptides and proteins encoded by a nucleic acid molecule can be the same or different.

In some embodiments, the encoded second polypeptide is also a reprogramming protein factor as described herein. In some embodiments, the encoded second polypeptide is selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, Lmx1b, or a functional derivative thereof. In some embodiments, the encoded second polypeptide is two or more polypeptides each independently selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, Lmx1b, or a functional derivative thereof.

In specific embodiments, the nucleic acid of the present disclosure comprises a bi-cistronic expression sequence encoding a NeuroD1 polypeptide and a Dlx2 polypeptide. In specific embodiments, the nucleic acid of the present disclosure comprises a bi-cistronic expression sequence encoding a NeuroD1 polypeptide and a Isl1 polypeptide. In specific embodiments, the nucleic acid of the present disclosure comprises a bi-cistronic expression sequence encoding a NeuroD1 polypeptide and a Ascl1 polypeptide. In some embodiments, the nucleic acid of the present disclosure comprises a bi-cistronic expression sequence encoding a NeuroD1 polypeptide and a NGN2 polypeptide. In some embodiments, the nucleic acid of the present disclosure comprises a bi-cistronic expression sequence encoding a NeuroD1 polypeptide and a Ctip2 polypeptide. In some embodiments, the nucleic acid of the present disclosure comprises a bi-cistronic expression sequence encoding a NeuroD1 polypeptide and a Math5 polypeptide. In some embodiments, the nucleic acid of the present disclosure comprises a bi-cistronic expression sequence encoding a NeuroD1 polypeptide and a Brn3a polypeptide. In some embodiments, the nucleic acid of the present disclosure comprises a bi-cistronic expression sequence encoding a NeuroD1 polypeptide and a Isl1 polypeptide. In specific embodiments, the nucleic acid of the present disclosure comprises a tri-cistronic expression sequence encoding a NeuroD1 polypeptide, a Dlx2 polypeptide, and Isl1 polypeptide. In specific embodiments, the nucleic acid of the present disclosure comprises a tri-cistronic expression sequence encoding a NeuroD1 polypeptide, a Dlx2 polypeptide, and Ascl1 polypeptide. In specific embodiments, the nucleic acid of the present disclosure comprises a tri- cistronic expression sequence encoding a NeuroD1 polypeptide, a Isl1 polypeptide, and Lhx3 polypeptide. In specific embodiments, the nucleic acid of the present disclosure comprises a tri-cistronic expression sequence encoding a NeuroD1 polypeptide, a Dlx2 polypeptide, and Ctip2 polypeptide. In specific embodiments, the nucleic acid of the present disclosure comprises a tri-cistronic expression sequence encoding a NeuroD1 polypeptide, a Ascl1 polypeptide, and Math5polypeptide. In specific embodiments, the nucleic acid of the present disclosure comprises a tri-cistronic expression sequence encoding a NeuroD1 polypeptide, a Ascl1 polypeptide, and Brn3a. In specific embodiments, the nucleic acid of the present disclosure comprises a tri-cistronic expression sequence encoding a NeuroD1 polypeptide, a Ascl1 polypeptide, and Isl1. In specific embodiments, the nucleic acid of the present disclosure comprises a tri-cistronic expression sequence encoding a NeuroD1 polypeptide, a Ascl1 polypeptide, and a Ctip2 polypeptide.

In specific embodiments, the nucleic acid of the present disclosure comprises a tetra-cistronic expression sequence encoding a NeuroD1 polypeptide, a Ascl1 polypeptide, a LMXIA polypeptide, and miR218 microRNA. In specific embodiments, the nucleic acid of the present disclosure comprises a tetra-cistronic expression sequence encoding a NeuroD1 polypeptide, a Ascl1 polypeptide, a Dlx2 polypeptide, and Ctip2 polypeptide. In specific embodiments, the nucleic acid of the present disclosure comprises a tetra-cistronic expression sequence encoding a NeuroD1 polypeptide, a Isl1 polypeptide, a Lhx3 polypeptide, and Dlx2 polypeptide. In specific embodiments, the nucleic acid of the present disclosure comprises a tetra-cistronic expression sequence encoding a NeuroD1 polypeptide, a Ascl1 polypeptide, a Dlx2 polypeptide, and Isl1 polypeptide.

In particular embodiments, the encoded Dlx2 polypeptide is a wild-type Dlx2. In some embodiments, the encoded Dlx2 is human Dlx2 having the amino acid sequence of SEQ ID NO: 3. In alternative embodiments, the encoded Dlx2 polypeptide is a functional derivative of Dlx2. In some embodiments, a functional derivative of Dlx2 shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) Dlx2 protein from which it derives. In some embodiments, the wild-type Dlx2 protein from which the functional derivative is derived is a wild-type human Dlx2 having SEQ ID NO: 3.

In some embodiments, the encoded Dlx2 polypeptide is encoded by (a) a DNA sequence of SEQ ID NO: 18, (b) a codon-optimized variant of (a) , or (c) a transcribed RNA sequence of (a) or (b) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 18. In some embodiments, the transcribed RNA sequence has the same sequence as the DNA coding sequences except that thymine bases in the DNA sequence are replaced by uracil bases in the RNA sequence.

In some embodiments, the encoded Dlx2 polypeptide is encoded by (a) a RNA sequence of SEQ ID NO: 19, or (b) a codon-optimized variant of (a) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 19.

In particular embodiments, the encoded Isl1 polypeptide is a wild-type Isl1. In some embodiments, the encoded Isl1 is human Isl1 having the amino acid sequence of SEQ ID NO: 4. In alternative embodiments, the encoded Isl1 polypeptide is a functional derivative of Isl1. In some embodiments, a functional derivative of Isl1 shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) Isl1 protein from which it derives. In some embodiments, the wild-type Isl1 protein from which the functional derivative is derived is a wild-type human Isl1 having SEQ ID NO: 4.

In some embodiments, the encoded Isl1 polypeptide is encoded by (a) a DNA sequence of SEQ ID NO: 20, (b) a codon-optimized variant of (a) , or (c) a transcribed RNA sequence of (a) or (b) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 20. In some embodiments, the transcribed RNA sequence has the same sequence as the DNA coding sequences except that thymine bases in the DNA sequence are replaced by uracil bases in the RNA sequence.

In some embodiments, the encoded Isl1 polypeptide is encoded by (a) a RNA sequence of SEQ ID NO: 21, or (b) a codon-optimized variant of (a) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 21.

In particular embodiments, the encoded Ascl1 polypeptide is a wild-type Ascl1. In some embodiments, the encoded Ascl1 is human Ascl1 having the amino acid sequence of SEQ ID NO: 5. In alternative embodiments, the encoded Ascl1 polypeptide is a functional derivative of Ascl1. In some embodiments, a functional derivative of Ascl1 shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to the native (e.g., wild-type) Ascl1 protein from which it derives. In some embodiments, the wild-type Ascl1 protein from which the functional derivative is derived is a wild-type human Ascl1 having SEQ ID NO: 5.

In some embodiments, the encoded Ascl1 polypeptide is encoded by (a) a DNA sequence of SEQ ID NO: 22, (b) a codon-optimized variant of (a) , or (c) a transcribed RNA sequence of (a) or (b) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 22. In some embodiments, the transcribed RNA sequence has the same sequence as the DNA coding sequences except that thymine bases in the DNA sequence are replaced by uracil bases in the RNA sequence.

In some embodiments, the encoded Ascl1 polypeptide is encoded by (a) a RNA sequence of SEQ ID NO: 23, or (b) a codon-optimized variant of (a) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 23.

In particular embodiments, the encoded Lhx3 polypeptide is a wild-type Lhx3. In some embodiments, the encoded Lhx3 is human Lhx3 having the amino acid sequence of SEQ ID NO: 6. In alternative embodiments, the encoded Lhx3 polypeptide is a functional derivative of Lhx3. In some embodiments, a functional derivative of Lhx3 shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) Lhx3 protein from which it derives. In some embodiments, the wild-type Lhx3 protein from which the functional derivative is derived is a wild-type human Lhx3 having SEQ ID NO: 6.

In some embodiments, the encoded Lhx3 polypeptide is encoded by (a) a DNA sequence of SEQ ID NO: 24, (b) a codon-optimized variant of (a) , or (c) a transcribed RNA sequence of (a) or (b) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 24. In some embodiments, the transcribed RNA sequence has the same sequence as the DNA coding sequences except that thymine bases in the DNA sequence are replaced by uracil bases in the RNA sequence.

In some embodiments, the encoded Lhx3 polypeptide is encoded by (a) a RNA sequence of SEQ ID NO: 25, or (b) a codon-optimized variant of (a) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 25.

In particular embodiments, the encoded Ngn2 polypeptide is a wild-type Ngn2. In some embodiments, the encoded Ngn2 is human Ngn2 having the amino acid sequence of SEQ ID NO: 7. In alternative embodiments, the encoded Ngn2 polypeptide is a functional derivative of Ngn2. In some embodiments, a functional derivative of Ngn2 shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) Ngn2 protein from which it derives. In some embodiments, the wild-type Ngn2 protein from which the functional derivative is derived is a wild-type human Ngn2 having SEQ ID NO: 7.

In some embodiments, the encoded Ngn2 polypeptide is encoded by (a) a DNA sequence of SEQ ID NO: 26, (b) a codon-optimized variant of (a) , or (c) a transcribed RNA sequence of (a) or (b) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 26. In some embodiments, the transcribed RNA sequence has the same sequence as the DNA coding sequences except that thymine bases in the DNA sequence are replaced by uracil bases in the RNA sequence.

In some embodiments, the encoded Ngn2 polypeptide is encoded by (a) a RNA sequence of SEQ ID NO: 27, or (b) a codon-optimized variant of (a) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 27.

In particular embodiments, the encoded LMX1A polypeptide is a wild-type LMX1A. In some embodiments, the encoded LMX1A is human LMX1A having the amino acid sequence of SEQ ID NO: 8. In alternative embodiments, the encoded LMX1A polypeptide is a functional derivative of LMX1A. In some embodiments, a functional derivative of LMX1A shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) LMX1A protein from which it derives. In some embodiments, the wild-type LMX1A protein from which the functional derivative is derived is a wild-type human LMX1A having SEQ ID NO: 8.

In some embodiments, the encoded LMX1A polypeptide is encoded by (a) a DNA sequence of SEQ ID NO: 28, (b) a codon-optimized variant of (a) , or (c) a transcribed RNA sequence of (a) or (b) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 28. In some embodiments, the transcribed RNA sequence has the same sequence as the DNA coding sequences except that thymine bases in the DNA sequence are replaced by uracil bases in the RNA sequence.

In some embodiments, the encoded LMX1A polypeptide is encoded by (a) a RNA sequence of SEQ ID NO: 29, or (b) a codon-optimized variant of (a) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 29.

In particular embodiments, the encoded Ctip2 polypeptide is a wild-type Ctip2. In some embodiments, the encoded Ctip2 is human Ctip2 having the amino acid sequence of SEQ ID NO: 9. In alternative embodiments, the encoded Ctip2 polypeptide is a functional derivative of Ctip2. In some embodiments, a functional derivative of Ctip2 shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) Ctip2 protein from which it derives. In some embodiments, the wild-type Ctip2 protein from which the functional derivative is derived is a wild-type human Ctip2 having SEQ ID NO: 9.

In some embodiments, the encoded Ctip2 polypeptide is encoded by (a) a DNA sequence of SEQ ID NO: 30, (b) a codon-optimized variant of (a) , or (c) a transcribed RNA sequence of (a) or (b) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 30. In some embodiments, the transcribed RNA sequence has the same sequence as the DNA coding sequences except that thymine bases in the DNA sequence are replaced by uracil bases in the RNA sequence.

In some embodiments, the encoded Ctip2 polypeptide is encoded by (a) a RNA sequence of SEQ ID NO: 31, or (b) a codon-optimized variant of (a) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 31.

In particular embodiments, the encoded Math5 polypeptide is a wild-type Math5. In some embodiments, the encoded Math5 is human Math5 having the amino acid sequence of SEQ ID NO: 10. In alternative embodiments, the encoded Math5 polypeptide is a functional derivative of Math5. In some embodiments, a functional derivative of Math5 shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) Math5 protein from which it derives. In some embodiments, the wild-type Math5 protein from which the functional derivative is derived is a wild-type human Math5 having SEQ ID NO: 10.

In some embodiments, the encoded Math5 polypeptide is encoded by (a) a DNA sequence of SEQ ID NO: 32, (b) a codon-optimized variant of (a) , or (c) a transcribed RNA sequence of (a) or (b) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 32. In some embodiments, the transcribed RNA sequence has the same sequence as the DNA coding sequences except that thymine bases in the DNA sequence are replaced by uracil bases in the RNA sequence.

In some embodiments, the encoded Math5 polypeptide is encoded by (a) a RNA sequence of SEQ ID NO: 33, or (b) a codon-optimized variant of (a) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 33.

In particular embodiments, the encoded Brn3a polypeptide is a wild-type Brn3a. In some embodiments, the encoded Brn3a is human Brn3a having the amino acid sequence of SEQ ID NO: 11. In alternative embodiments, the encoded Brn3a polypeptide is a functional derivative of Brn3a. In some embodiments, a functional derivative of Brn3a shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) Brn3a protein from which it derives. In some embodiments, the wild-type Brn3a protein from which the functional derivative is derived is a wild-type human Brn3a having SEQ ID NO: 11.

In some embodiments, the encoded Brn3a polypeptide is encoded by (a) a DNA sequence of SEQ ID NO: 34, (b) a codon-optimized variant of (a) , or (c) a transcribed RNA sequence of (a) or (b) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 34. In some embodiments, the transcribed RNA sequence has the same sequence as the DNA coding sequences except that thymine bases in the DNA sequence are replaced by uracil bases in the RNA sequence.

In some embodiments, the encoded Brn3a polypeptide is encoded by (a) a RNA sequence of SEQ ID NO: 35, or (b) a codon-optimized variant of (a) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 35.

In some embodiments, the multi-cistronic expression sequence encoding the NeuroD1 polypeptide and at least one second polypeptide further comprises a non-coding sequence separating two ORFs comprises an internal ribosome entry site (IRES) . Without being bound by the theory, it is contemplated that an internal ribosome entry sites (IRES) can act as the sole ribosome binding site, or serve as one of multiple ribosome binding sites of an mRNA. An mRNA molecule containing more than one functional ribosome binding site can encode several peptides or proteins that are translated independently by the ribosomes (e.g., multicistronic mRNA) . Accordingly, in some embodiments, the nucleic acid molecule of the present disclosure (e.g., mRNA) comprises one or more internal ribosome entry sites (IRES) . Examples of IRES sequences that can be used in connection with the present disclosure include, without limitation, those from picomaviruses (e.g., FMDV) , pest viruses (CFFV) , polio viruses (PV) , encephalomyocarditis viruses (ECMV) , foot-and-mouth disease viruses (FMDV) , hepatitis C viruses (HCV) , classical swine fever viruses (CSFV) , murine leukemia virus (MLV) , simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV) .

In particular embodiments, the IRES has a sequence of an IRES from Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n. myc, Mouse Gtx, Human p27kip1, Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E/D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Apodemus Agrarius Picornavirus, Caprine Kobuvirus, Parabovirus, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or an aptamer to eIF4G.

Table 3 shows exemplary IRES sequences that can be used in connection with the present disclosure.

Table 3 Exemplary IRES sequences.

Additionally, various IRES sequences that are known in the art and can be used in connection with the present disclosure. For example, in some embodiments, the IRES can have a sequence disclosed in U.S. Patent Application Publication No.: 2022/0323480A1 and particularly an IRES sequence listed in Table 17, the content of which is incorporated by reference in its entirety.

Modifications of IRES and accessory sequences are disclosed herein to increase or reduce IRES activities, for example, by truncating the 5′ and/or 3′ ends of the IRES, adding a spacer 5′ to the IRES, modifying the 6 nucleotides 5′ to the translation initiation site (Kozak sequence) , modification of alternative translation initiation sites, and creating chimeric/hybrid IRES sequences. In some embodiments, the IRES sequence in the circular RNA disclosed herein comprises one or more of these modifications relative to a native IRES. In some embodiments, the IRES sequence comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an IRES sequence in Table 3.

In some embodiments, the multi-cistronic expression sequence encoding the NeuroD1 polypeptide and at least one second polypeptide further comprises a ribosomal skipping element that separates the coding sequences for two encoded polypeptides. In some embodiments, the ribosomal skipping element can terminate translation of the first polypeptide chain and re-initiating translation of the second polypeptide chain from the nucleic acid molecule. In alternative embodiments, the ribosomal skipping element encodes a protease cleavage site in the polypeptide encoded by the nucleic acid molecule, so that the polypeptide can be cleaved by an intrinsic protease activity of its own, or by another protease in its environment to produce two polypeptide chains. In specific embodiments, the ribosomal skipping element encodes thosea-asigna virus 2A peptide (T2A) , porcine teschovirus-1 2 A peptide (P2A) , foot-and-mouth disease virus 2 A peptide (F2A) , equine rhinitis A vims 2A peptide (E2A) , cytoplasmic polyhedrosis vims 2A peptide (BmCPV 2A) , or flacherie vims of B. mori 2A peptide (BmIFV 2A) . Table 4 shows exemplary sequences of the ribosomal skipping element.

Table 4 Exemplary sequences of the ribosomal skipping element.

5.3.2 Untranslated Regions (UTRs)

In some embodiments, the nucleic acid molecules of the present disclosure comprise one or more untranslated regions (UTRs) .

In the particular embodiments, where the functional nucleic acid molecule is a linear molecule, the untranslated region (UTR) located upstream (to the 5’-end) of the coding region is referred to herein as the 5’-UTR, and the UTR located upstream (to the 3’-end) of the coding region is referred to herein as the 3’-UTR. In particular embodiments, the nucleic acid molecule comprises both a 5’-UTR and a 3’-UTR. In some embodiments, the 5’-UTR comprises a 5’-Cap structure as described herein. In some embodiments, the nucleic acid molecule comprises a Kozak sequence (e.g., in the 5’-UTR) . In some embodiments, the nucleic acid molecule comprises a poly-A region (e.g., in the 3’-UTR) . In some embodiments, the nucleic acid molecule comprises a polyadenylation signal (e.g., in the 3’-UTR) . In some embodiments, the nucleic acid molecule comprises stabilizing region (e.g., in the 3’-UTR) . In some embodiments, the nucleic acid molecule comprises a secondary structure. In some embodiments, the secondary structure is a stem-loop. In some embodiments, the nucleic acid molecule comprises a stem-loop sequence (e.g., in the 5’-UTR and/or the 3’-UTR) . In some embodiments, the nucleic acid molecule comprises one or more intronic regions capable of being excised during splicing. In a specific embodiment, the nucleic acid molecule comprises one or more region selected from a 5’-UTR, and a coding region. In a specific embodiment, the nucleic acid molecule comprises one or more region selected from a coding region and a 3’-UTR. In a specific embodiment, the nucleic acid molecule comprises one or more region selected from a 5’-UTR, a coding region, and a 3’-UTR.

In the alternative embodiments, where the functional nucleic acid molecule is a circular molecule (e.g., a circular RNA molecule) , the UTR or UTRs can arrange in particular order with the coding sequence in the circular molecule.

In some embodiments, the sequence of an UTR can be homologous or heterologous to the sequence of the coding region found in a nucleic acid molecule. Multiple UTRs can be included in a nucleic acid molecule and can be of the same or different sequences, and/or genetic origin. According to the present disclosure, any portion of UTRs in a nucleic acid molecule (including none) can be codon optimized and any may independently contain one or more different structural or chemical modification, before and/or after codon optimization.

In some embodiments, a nucleic acid molecule of the present disclosure (e.g., linear RNA or circular RNA) comprises UTRs and coding regions that are homologous with respect to each other. In other embodiments, a nucleic acid molecule of the present disclosure (e.g., linear RNA or circular RNA) comprises UTRs and coding regions that are heterologous with respect to each other. In some embodiments, to monitor the activity of a UTR sequence, a nucleic acid molecule comprising the UTR and a coding sequence of a detectable probe can be administered in vitro (e.g., cell or tissue culture) or in vivo (e.g., to a subject) , and an effect of the UTR sequence (e.g., modulation on the expression level, cellular localization of the encoded product, or half-life of the encoded product) can be measured using methods known in the art.

In some embodiments, a nucleic acid molecule of the present disclosure (e.g., linear RNA or circular RNA) comprises at least one internal ribosome entry sites (IRES) in its UTR, and the IRES is operably linked to a coding sequence. In some embodiments, the coding sequence is as described in Section 5.3.1 (coding region) . Examples of IRES sequences that can be used in connection with the present disclosure include, without limitation, those from picomaviruses (e.g., FMDV) , pest viruses (CFFV) , polio viruses (PV) , encephalomyocarditis viruses (ECMV) , foot-and-mouth disease viruses (FMDV) , hepatitis C viruses (HCV) , classical swine fever viruses (CSFV) , murine leukemia virus (MLV) , simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV) .

In specific embodiments, the circular RNA or the linear precursor RNA comprises an IRES having the sequence as shown in Table 3 or a transcribed RNA sequence thereof in at least one of its UTR. In some embodiments, the transcribed RNA sequence has the same sequence as the DNA sequence coding for such IRES, except that thymine bases in the DNA sequence are replaced by uracil bases in the RNA sequence.

In some embodiments, the UTR of a nucleic acid molecule of the present disclosure (e.g., linear RNA or circular RNA) comprises at least one translation enhancer element (TEE) that functions to increase the amount of polypeptide or protein produced from the nucleic acid molecule. In some embodiments where the nucleic acid molecule is linear, the TEE is located in the 5’-UTR of the nucleic acid molecule. In other embodiments, the TEE is located at the 3’-UTR of the nucleic acid molecule. In yet other embodiments, at least two TEE are located at the 5’-UTR and 3’-UTR of the nucleic acid molecule respectively. In some embodiments, a nucleic acid molecule of the present disclosure (e.g., mRNA) can comprise one or more copies of a TEE sequence or comprise more than one different TEE sequences. In some embodiments, different TEE sequences that are present in a nucleic acid molecule of the present disclosure can be homologues or heterologous with respect to one another.

In some embodiments, the TEE sequence is derived from a promoter sequence of a gene. In some embodiments, a promoter can be derived entirely from a single gene. In other embodiments, a promoter can be chimeric, having portions derived from more than one gene.

In some embodiments, the TEE sequence used in connection with the present disclosure can drive expression of an operably linked expression sequence preferentially in glial cells. In some embodiments, the TEE sequence drives expression of an operably linked expression sequence preferentially in astrocytes. In some embodiments, the TEE sequence drives expression of an operably linked expression sequence preferentially in reactive astrocytes. In some embodiments, the TEE sequence drives expression of an operably linked expression sequence preferentially in NG2 cells. In some embodiments, the TEE sequence drives expression of an operably linked expression sequence preferentially in reactive NG2 cells. In some embodiments, the TEE sequence drives expression of an operably linked expression sequence preferentially in Müller glia cells.

Additionally, various TEE sequences that are known in the art and can be used in connection with the present disclosure. For example, in some embodiments, the TEE can be an internal ribosome entry site (IRES) , HCV-IRES or an IRES element. Chappell et al. Proc. Natl. Acad. Sci. USA 101: 9590-9594, 2004; Zhou et al. Proc. Natl. Acad. Sci. 102: 6273-6278, 2005. Additional internal ribosome entry site (IRES) that can be used in connection with the present disclosure include but are not limited to those described in U.S. Patent No. 7,468,275, U.S. Patent Publication No. 2007/0048776 and U.S. Patent Publication No. 2011/0124100 and International Patent Publication No. WO2007/025008 and International Patent Publication No. WO2001/055369, the content of each of which is enclosed herein by reference in its entirety. In some embodiments, the TEE can be those described in Supplemental Table 1 and in Supplemental Table 2 of Wellensiek et al Genome-wide profiling of human cap-independent translation-enhancing elements, Nature Methods, 2013 Aug; 10 (8) : 747–750; the content of which is incorporated by reference in its entirety.

Additional exemplary TEEs that can be used in connection with the present disclosure include but are not limited to the TEE sequences disclosed in U.S. Patent No. 6,310,197, U.S. Patent No. 6,849,405, U.S. Patent No. 7,456,273, U.S. Patent No. 7,183,395, U.S. Patent Publication No. 2009/0226470, U.S. Patent Publication No. 2013/0177581, U.S. Patent Publication No. 2007/0048776, U.S. Patent Publication No. 2011/0124100, U.S. Patent Publication No. 2009/0093049, International Patent Publication No. WO2009/075886, International Patent Publication No. WO2012/009644, and International Patent Publication No. WO1999/024595, International Patent Publication No.WO2007/025008, International Patent Publication No. WO2001/055371, European Patent No. 2610341, European Patent No. 2610340, the content of each of which is enclosed herein by reference in its entirety.

Combinations of regulatory sequences may be used to drive expression of an operably linked expression sequence. According to the present disclosure, homologues and functional variants of ubiquitous or cell type-specific promoters may be used in expressing the operably linked expression sequence as described herein. The terms “promoter homologue” and “promoter variant” refer to a promoter which has substantially similar functional properties to confer the desired type of expression, such as cell type-specific expression of the NeuroD1 polypeptide or ubiquitous expression of the NeuroD1 polypeptide, of an operably linked nucleic acid encoding the NeuroD1 polypeptide compared to a given promoter disclosed herein. For example, a promoter homologue or promoter variant has substantially similar functional properties to confer cell type-specific expression of an operably linked nucleic acid encoding the NeuroD1 polypeptide compared to any of a GFAP, AldhlL1, NG2, lcn2, S100b, Sox9, CAG, CMV, ubiquitin, or EF-1a promoter.

One of skill in the art will recognize that one or more nucleic acid mutations can be introduced without altering the functional properties of a given promoter. Mutations can be introduced using standard molecular biology techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis, to produce promoter variants. As used herein, the term “promoter variant” refers to either an isolated naturally occurring or a recombinantly prepared variation of a reference promoter, such as, but not limited to GFAP, AldhlL1, NG2, lcn2, S100b, Sox9, CAG, CMV, ubiquitin, or EF-1a promoter.

It is known in the art that promoters from other species are functional, e.g. the mouse AldhlLl promoter is known to be functional in human cells. Homologues and homologous promoters from other species can be identified using bioinformatics tools known in the art, see for example, Xuan et al., 2005, Genome Biol 6: R72; Zhao et al., 2005, Nucl Acid Res 33: D103-107; and Halees et al. 2003, Nucl. Acids. Res. 2003 31: 3554-3559.

Structurally, homologues and variants of a cell type-specific promoter or an ubiquitous promoter can have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, nucleic acid sequence identity to the reference promoter and include a site for binding of RNA polymerase and, optionally, one or more binding sites for transcription factors.

In various embodiments, a nucleic acid molecule of the present disclosure (e.g., linear RNA or circular RNA) comprises at least one UTR that comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences. In some embodiments, the TEE sequences in the UTR of a nucleic acid molecule are copies of the same TEE sequence. In other embodiments, at least two TEE sequences in the UTR of a nucleic acid molecule are of different TEE sequences. In some embodiments, multiple different TEE sequences are arranged in one or more repeating patterns in the UTR region of a nucleic acid molecule. For illustrating purpose only, a repeating pattern can be, for example, ABABAB, AABBAABBAABB, ABCABCABC, or the like, where in these exemplary patterns, each capitalized letter (A, B, or C) represents a different TEE sequence. In some embodiments, at least two TEE sequences are consecutive with one another (i.e., no spacer sequence in between) in a UTR of a nucleic acid molecule. In other embodiments, at least two TEE sequences are separated by a spacer sequence. In some embodiments, a UTR can comprise a TEE sequence-spacer sequence module that is repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or more than 9 times in the UTR. In any of the embodiments described in this paragraph, the UTR can be a 5’-UTR, a 3’-UTR or both 5’-UTR and 3’-UTR of a nucleic acid molecule.

In some embodiments, the UTR of a nucleic acid molecule of the present disclosure (e.g., a linear RNA or circular RNA) comprises at least one translation suppressing element that functions to decrease the amount of polypeptide or protein produced from the nucleic acid molecule. In some embodiments, the UTR of the nucleic acid molecule comprises one or more miR sequences or fragment thereof (e.g., miR seed sequences) that are recognized by one or more microRNA. In some embodiments, the UTR of the nucleic acid molecule comprises one or more stem-loop structure that downregulates translational activity of the nucleic acid molecule. Other mechanisms for suppressing translational activities associated with a nucleic acid molecules are known in the art. In some of the embodiments described in this paragraph, the nucleic acid molecule is linear, and the UTR can be a 5’-UTR, a 3’-UTR or both 5’-UTR and 3’-UTR of a nucleic acid molecule.

Table 5 shows exemplary 5’-UTR and 3’-UTR sequences that can be operably linked to an expression sequence in a linear RNA molecule (e.g., linear mRNA) as described herein.

Table 5 Exemplary UTRs in linear molecules.

5.3.3 Circular Nucleic Acid Molecules

According to one aspect of the present disclosure, the functional nucleic acid molecules provided herein are, or are configured to produce, a circular nucleic acid molecule, and particularly a circular RNA molecule. Accordingly, provided herein are circular RNAs, precursor RNAs that can circularize into the circular RNAs, and vectors (e.g., DNA vectors) that can be transcribed into the precursor RNAs or the circular RNAs.

In some embodiments, provided herein is a linear precursor RNA molecule that is capable of forming a circular RNA through a ribozyme self-splicing reaction. In specific embodiments, a linear precursor RNA according to the present disclosure comprises in the following order:

(a) a 3’ group I intron fragment or an analog thereof;

(b) an IRES;

(d) a 5’ group I intron fragment or an analog thereof;

In some embodiments, provided herein is a circular RNA molecule that is formed by circularization of a linear precursor RNA molecule described herein through a ribozyme self-splicing reaction of the linear precursor RNA molecule.

In some embodiments, provided herein is a circular RNA molecule comprising, in the following order:

(a) a post-splicing 3’ group I intron fragment;

(b) an IRES;

(d) a post-splicing 5’ group I intron fragment.

In some embodiments, the expression sequence in the linear precursor RNA or the circular RNA is selected from an expression sequence described in Section 5.3.1 (coding sequence) . In particular embodiments, the expression sequence comprises (a) a DNA sequence of SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, (b) a codon-optimized variant of (a) , or (c) a transcribed RNA sequence of (a) or (b) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 12. In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 13. In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 14. In some embodiments, the transcribed RNA sequence has the same sequence as the DNA coding sequences except that thymine bases in the DNA sequence are replaced by uracil bases in the RNA sequence.

In some embodiments, the expression sequence comprises (a) an RNA sequence of NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, or (b) a codon-optimized variant of (a) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 15. In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 16. In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 17.

In some embodiments, the IRES sequence or the in the linear precursor RNA or the circular RNA is selected from an IRES from picomaviruses (e.g., FMDV) , pest viruses (CFFV) , polio viruses (PV) , encephalomyocarditis viruses (ECMV) , foot-and-mouth disease viruses (FMDV) , hepatitis C viruses (HCV) , classical swine fever viruses (CSFV) , murine leukemia virus (MLV) , simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV) .

In specific embodiments, the linear precursor RNA or the circular RNA comprises an IRES having the sequence as shown in Table 3 or a transcribed RNA sequence thereof. In some embodiments, the transcribed RNA sequence has the same sequence as the DNA sequence coding for such IRES, except that thymine bases in the DNA sequence are replaced by uracil bases in the RNA sequence. In particular embodiments, the linear precursor RNA molecule disclosed herein comprises an IRES sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an IRES sequence in Table 3.

In some embodiments, the linear precursor RNA comprises a 3’ group I intron or an analog thereof. Table 6 shows exemplary 3’ group I intron fragment sequences that can be included in a linear precursor RNA molecule as described herein.

Table 6 Exemplary 3’ group I intron fragment sequences.

In some embodiments, 3’ group I intron fragment comprises a sequence of SEQ ID NO: 61. In some embodiments, the 3’ group I intron fragment comprises a sequence of at least about 75%sequence identity to SEQ ID NO: 61. In some embodiments, the 3’ group I intron fragment comprises a sequence of at least about 80%sequence identity to SEQ ID NO: 61. In some embodiments, the 3’ group I intron fragment comprises a sequence of at least about 85%sequence identity to SEQ ID NO: 61. In some embodiments, the 3’ group I intron fragment comprises a sequence of at least about 90%sequence identity to SEQ ID NO: 61. In some embodiments, the 3’ group I intron fragment comprises a sequence of at least about 95%sequence identity to SEQ ID NO: 61. In some embodiments, the 3’ group I intron fragment comprises a sequence of at least about 97%sequence identity to SEQ ID NO: 61. In some embodiments, the 3’ group I intron fragment comprises a sequence of at least about 99%sequence identity to SEQ ID NO: 61.

Additionally, various 3’ group I intron fragment that are known in the art and can be used in connection with the present disclosure. For example, in some embodiments, the 3’ group I intron fragment can have a sequence disclosed in U.S. Patent Application Publication No.: 2022/0323480A1 and particularly a sequence listed in Table 19 therein, the content of which is incorporated by reference in its entirety.

In some embodiments, the linear precursor RNA comprises a 5’ group I intron or an analog thereof. Table 7 shows exemplary 5’ group I intron fragment sequences that can be included in a linear precursor RNA molecule as described herein.

Table 7 Exemplary 5’ group I intron fragment sequences.

In some embodiments 5’ group I intron fragment comprises a sequence of SEQ ID NO: 63. In some embodiments, the 5’ group I intron fragment comprises a sequence of at least about 75%sequence identity to SEQ ID NO: 63. In some embodiments, the 5’ group I intron fragment comprises a sequence of at least about 80%sequence identity to SEQ ID NO: 63. In some embodiments, the 5’ group I intron fragment comprises a sequence of at least about 85%sequence identity to SEQ ID NO: 63. In some embodiments, the 5’ group I intron fragment comprises a sequence of at least about 90%sequence identity to SEQ ID NO: 63. In some embodiments, the 5’ group I intron fragment comprises a sequence of at least about 95%sequence identity to SEQ ID NO: 63. In some embodiments, the 5’ group I intron fragment comprises a sequence of at least about 97%sequence identity to SEQ ID NO: 63. In some embodiments, the 5’ group I intron fragment comprises a sequence of at least about 99%sequence identity to SEQ ID NO: 63.

Additionally, various 5’ group I intron fragment that are known in the art and can be used in connection with the present disclosure. For example, in some embodiments, the 5’ group I intron fragment can have a sequence disclosed in U.S. Patent Application Publication No.: 2022/0323480A1 and particularly a sequence listed in Table 18 therein, the content of which is incorporated by reference in its entirety.

In some embodiments, the circular RNA comprises a post-splicing 3’ group I intron or an analog thereof. Table 8 shows exemplary post-splicing 3’ group I intron fragment sequences that can be included in circular RNA molecule as described herein.

Table 8 Exemplary post-splicing 3’ group I intron fragment sequences.

In some embodiments a post-splicing 3’ group I intron fragment comprises a sequence of SEQ ID NO: 64. In some embodiments, the post-splicing 3’ group I intron fragment comprises a sequence of at least about 75%sequence identity to SEQ ID NO: 64. In some embodiments, the post-splicing 3’ group I intron fragment comprises a sequence of at least about 80%sequence identity to SEQ ID NO: 64. In some embodiments, the post-splicing 3’ group I intron fragment comprises a sequence of at least about 85%sequence identity to SEQ ID NO: 64. In some embodiments, the post-splicing 3’ group I intron fragment comprises a sequence of at least about 90%sequence identity to SEQ ID NO: 64. In some embodiments, the post-splicing 3’ group I intron fragment comprises a sequence of at least about 95%sequence identity to SEQ ID NO: 64. In some embodiments, the post-splicing 3’ group I intron fragment comprises a sequence of at least about 97%sequence identity to SEQ ID NO: 64. In some embodiments, the post-splicing 3’ group I intron fragment comprises a sequence of at least about 99%sequence identity to SEQ ID NO: 64.

In some embodiments, the circular RNA comprises a post-splicing 5’ group I intron or an analog thereof. Table 9 shows exemplary post-splicing 5’ group I intron fragment sequences that can be included in circular RNA molecule as described herein.

Table 9 Exemplary post-splicing 5’ group I intron fragment sequences.

In some embodiments a post-splicing 5’ group I intron fragment comprises a sequence of SEQ ID NO: 65. In some embodiments, the post-splicing 5’ group I intron fragment comprises a sequence of at least about 75%sequence identity to SEQ ID NO: 65. In some embodiments, the post-splicing 5’ group I intron fragment comprises a sequence of at least about 80%sequence identity to SEQ ID NO: 65. In some embodiments, the post-splicing 5’ group I intron fragment comprises a sequence of at least about 85%sequence identity to SEQ ID NO: 65. In some embodiments, the post-splicing 5’ group I intron fragment comprises a sequence of at least about 90%sequence identity to SEQ ID NO: 65. In some embodiments, the post-splicing 5’ group I intron fragment comprises a sequence of at least about 95%sequence identity to SEQ ID NO: 65. In some embodiments, the post-splicing 5’ group I intron fragment comprises a sequence of at least about 97%sequence identity to SEQ ID NO: 65. In some embodiments, the post-splicing 5’ group I intron fragment comprises a sequence of at least about 99%sequence identity to SEQ ID NO: 65.

In some embodiments, the linear precursor RNA further comprises one or more spacer sequences. Two types of spacers have been designed for improving precursor RNA circularization and/or gene expression from circular RNA. The first type of spacer is external spacer, i.e., present in a precursor RNA but removed upon circularization. While not wishing to be bound by theory, it is contemplated that an external spacer may improve ribozyme-mediated circularization by maintaining the structure of the ribozyme itself and preventing other neighboring sequence elements from interfering with its folding and function. The second type of spacer is internal spacer, i.e., present in a precursor RNA and retained in a resulting circular RNA. While not wishing to be bound by theory, it is contemplated that an internal spacer may improve ribozyme-mediated circularization by maintaining the structure of the ribozyme itself and preventing other neighboring sequence elements, particularly the neighboring IRES and coding region, from interfering with its folding and function. It is also contemplated that an internal spacer may improve protein expression from the IRES by preventing neighboring sequence elements, particularly the intron elements, from hybridizing with sequences within the IRES and inhibiting its ability to fold into its most preferred and active conformation.

In certain aspects, provided herein are circular RNA polynucleotides comprising a 3′ post splicing group I intron fragment, optionally a first spacer, an Internal Ribosome Entry Site (IRES) , an expression sequence, optionally a second spacer, and a 5′ post splicing group I intron fragment. In some embodiments, these regions are in that order. In some embodiments, the circular RNA is made by a method provided herein or from a vector provided herein.

In some embodiments, the vectors and linear precursor RNA polynucleotides provided herein comprise a first (5′) external complementary sequence and a second (3′) external complementary sequence. In certain embodiments, the first and second external complementary sequences may form perfect or imperfect duplexes. Thus, in certain embodiments at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%of the first and second external complementary sequences may be base paired with one another. In some embodiments, the complementary sequences are predicted to have less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%) base pairing with unintended sequences in the RNA (e.g., a sequence outside the complementary sequences) . In some embodiments, including such external complementary sequences on the ends of the precursor RNA strand, and adjacent or very close to the group I intron fragment, bring the group I intron fragments in close proximity to each other, increasing splicing efficiency. In some embodiments, the external complementary sequences are 3 to 100 nucleotides in length (e.g., 3-75 nucleotides in length, 3-50 nucleotides in length, 20-50 nucleotides in length, 35-50 nucleotides in length, 5-25 nucleotides in length, 9-19 nucleotides in length) . In some embodiments, the external complementary sequences are about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In some embodiments, the external complementary sequences have a length of about 9 to about 50 nucleotides. In one embodiment, the external complementary sequences have a length of about 9 to about 19 nucleotides. In some embodiments, the external complementary sequences have a length of about 20 to about 40 nucleotides. In certain embodiments, the external complementary sequences have a length of about 30 nucleotides.

In certain embodiments, the vectors, precursor RNA and circular RNA provided herein comprise a first (5′) and/or a second (3′) spacer. In some embodiments, including a spacer between the 3′ group I intron fragment and the IRES may conserve secondary structures in those regions by preventing them from interacting, thus increasing splicing efficiency. In some embodiments, the vectors, precursor RNA and circular RNA provided herein comprises a first (between 3′ group I intron fragment and IRES) and second (between the expression sequence and 5′ group I intron fragment) internal complementary sequences comprise additional base pairing regions that are predicted to base pair with each other and not to the first and second external complementary sequences. In some embodiments, such base pairing between the first and second internal complementary sequences brings the group I intron fragments in close proximity to each other, further increasing splicing efficiency. Additionally, in some embodiments, the combination of base pairing between the first and second external complementary sequences, and separately, base pairing between the first and second internal complementary sequence, promotes the formation of a splicing bubble containing the group I intron fragments flanked by adjacent regions of base pairing.

Typical spacers are contiguous sequences with one or more of the following qualities: 1) predicted to avoid interfering with proximal structures, for example, the IRES, expression sequence, or intron; 2) is at least 7 nt long and no longer than 100 nt; 3) is located after and adjacent to the 3′ intron fragment and/or before and adjacent to the 5′ intron fragment; and 4) contains one or more of the following: a) an unstructured region at least 5 nt long, b) a region of base pairing at least 5 nt long to a distal sequence, including another spacer, and c) a structured region at least 7 nt long limited in scope to the sequence of the spacer. Spacers may have several regions, including an unstructured region, a base pairing region, a hairpin/structured region, and combinations thereof. In an embodiment, the spacer has a structured region with high GC content. In an embodiment, a region within a spacer base pairs with another region within the same spacer. In an embodiment, a region within a spacer base pairs with a region within another spacer. In an embodiment, a spacer comprises one or more hairpin structures. In an embodiment, a spacer comprises one or more hairpin structures with a stem of 4 to 12 nucleotides and a loop of 2 to 10 nucleotides. In an embodiment, there is an additional spacer between the 3′ group I intron fragment and the IRES. In an embodiment, this additional spacer prevents the structured regions of the IRES from interfering with the folding of the 3′ group I intron fragment or reduces the extent to which this occurs. In some embodiments, the 5′ spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the 5′ spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 5′ spacer sequence is between 5 and 50, 10 and 50, 20 and 50, 20 and 40, and/or 25 and 35 nucleotides in length. In certain embodiments, the 5′ spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In one embodiment, the 5′ spacer sequence is a polyA sequence. In another embodiment, the 5′ spacer sequence is a polyAC sequence. In one embodiment, a spacer comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%polyAC content. In one embodiment, a spacer comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%polypyrimidine (C/T or C/U) content.

In certain embodiments, a circular RNA polynucleotide provided herein comprises, in the following order, a 3′ post splicing group I intron fragment, a first internal spacer, an Internal Ribosome Entry Site (IRES) , an expression sequence, a second internal spacer, and a 5′ post splicing group I intron fragment. In some embodiments, the first internal spacer is about 10 to about 80 nucleotides long. In some embodiments, the first internal spacer is about 15 to about 45 nucleotides long. In some embodiments, the first internal spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55 or about 60 nucleotides long. In some embodiments, the second internal spacer is about 10 to about 80 nucleotides long. In some embodiments, the second internal spacer is about 15 to about 45 nucleotides long. In some embodiments, the second internal spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55 or about 60 nucleotides long.

In certain embodiments, a circular RNA polynucleotide provided herein comprises, in the following order, a 3′ post splicing group I intron fragment, a first internal complementary sequence, a first internal spacer, an Internal Ribosome Entry Site (IRES) , an expression sequence, a second internal spacer, a second internal complementary sequence, and a 5′ post splicing group I intron fragment, wherein the first and second internal complementary sequences have about 85%to about 100%complementarity when read in the opposite directions of one another. In some embodiments, the first and second internal complementary sequences have about 85%, about 90%, about 95%, about 98%, or about 100%complementarity when read in the opposite directions of one another. In some embodiments, the first internal spacer is about 10 to about 80 nucleotides long. In some embodiments, the first internal spacer is about 15 to about 45 nucleotides long. In some embodiments, the first internal spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, or about 80 nucleotides long. In some embodiments, the second internal spacer is about 10 to about 80 nucleotides long. In some embodiments, the second internal spacer is about 15 to about 45 nucleotides long. In some embodiments, the second internal spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75 or about 80 nucleotides long.

In certain embodiments, transcription of a vector provided herein results in the formation of a precursor linear RNA polynucleotide capable of circularizing. In some embodiments, this precursor linear RNA polynucleotide circularizes when incubated in the presence of guanosine nucleotide or nucleoside (e.g., GTP) and divalent cation (e.g., Mg²⁺) . In some embodiments, the precursor linear RNA polynucleotide comprises, from 5’ to 3’, a 3′ group I intron fragment, optionally a first spacer, an Internal Ribosome Entry Site (IRES) , an expression sequence, optionally a second spacer, and a 5′ group I intron fragment.

In certain embodiments, the precursor linear RNA polynucleotide comprises, from 5’ to 3’, a 3′ group I intron fragment, a first internal spacer, an Internal Ribosome Entry Site (IRES) , an expression sequence, a second internal spacer, and a 5′ group I intron fragment. In some embodiments, the first internal spacer is about 10 to about 80 nucleotides long. In some embodiments, the first internal spacer is about 15 to about 45 nucleotides long. In some embodiments, the first internal spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, or about 80 nucleotides long. In some embodiments, the second internal spacer is about 10 to about 80 nucleotides long. In some embodiments, the second internal spacer is about 15 to about 45 nucleotides long. In some embodiments, the second internal spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, or about 80 nucleotides long.

In certain embodiments, the precursor linear RNA polynucleotide provided herein comprises, from 5’ to 3’, a 3′ group I intron fragment, a first internal complementary sequence, a first internal spacer, an Internal Ribosome Entry Site (IRES) , an expression sequence, a second internal spacer, a second internal complementary sequence, and a 5′ group I intron fragment, wherein the first and second internal complementary sequences have about 85%to about 100%complementarity when read in the opposite directions of one another. In some embodiments, the first and second internal complementary sequences have about 85%, about 90%, about 95%, about 98%, or about 100%complementarity when read in the opposite directions of one another. In some embodiments, the first internal spacer is about 10 to about 80 nucleotides long. In some embodiments, the first internal spacer is about 15 to about 45 nucleotides long. In some embodiments, the first internal spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55 or about 60 nucleotides long. In some embodiments, the second internal spacer is about 10 to about 80 nucleotides long. In some embodiments, the second internal spacer is about 15 to about 45 nucleotides long. In some embodiments, the second internal spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, or about 80 nucleotides long.

In certain embodiments, the precursor linear RNA polynucleotide provided herein comprises, from 5’ to 3’, a 3′ group I intron fragment, a first external spacer, a first internal complementary sequence, a first internal spacer, an Internal Ribosome Entry Site (IRES) , an expression sequence, a second internal spacer, a second internal complementary sequence, a second external spacer, and a 5′ group I intron fragment, wherein the first and second internal complementary sequences have about 85%to about 100%complementarity when read in the opposite directions of one another. In some embodiments, the first and second internal complementary sequences have about 85%, about 90%, about 95%, about 98%, or about 100%complementarity when read in the opposite directions of one another. In some embodiments, the first internal spacer is about 10 to about 80 nucleotides long. In some embodiments, the first internal spacer is about 15 to about 45 nucleotides long. In some embodiments, the first internal spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, or about 80 nucleotides long. In some embodiments, the second internal spacer is about 10 to about 80 nucleotides long. In some embodiments, the second internal spacer is about 15 to about 45 nucleotides long. In some embodiments, the second internal spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, or about 80 nucleotides long. In some embodiments, the first external spacer is about 10 to about 80 nucleotides long. In some embodiments, the first external spacer is about 15 to about 45 nucleotides long. In some embodiments, the first external spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75 or about 80 nucleotides long. In some embodiments, the second external spacer is about 10 to about 80 nucleotides long. In some embodiments, the second external spacer is about 15 to about 45 nucleotides long. In some embodiments, the second external spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75 or about 80 nucleotides long.

In certain embodiments, the precursor linear RNA polynucleotide provided herein comprises, from 5’ to 3’, a first external complementary sequence, a 3′ group I intron fragment, a first external spacer, a first internal complementary sequence, a first internal spacer, an Internal Ribosome Entry Site (IRES) , an expression sequence, a second internal spacer, a second internal complementary sequence, a second external spacer, and a 5′ group I intron fragment, and a second external complementary sequence. In some embodiments, the first and second internal complementary sequences have about 85%to about 100%complementarity when read in the opposite directions of one another. In some embodiments, the first and second internal complementary sequences have about 85%, about 90%, about 95%, about 98%, or about 100%complementarity when read in the opposite directions of one another. In some embodiments, the first and second external complementary sequences have about 85%to about 100%complementarity when read in the opposite directions of one another. In some embodiments, the first and second external complementary sequences have about 85%, about 90%, about 95%, about 98%, or about 100%complementarity when read in the opposite directions of one another. In some embodiments, the first internal spacer is about 10 to about 80 nucleotides long. In some embodiments, the first internal spacer is about 15 to about 45 nucleotides long. In some embodiments, the first internal spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, or about 80 nucleotides long. In some embodiments, the second internal spacer is about 10 to about 80 nucleotides long. In some embodiments, the second internal spacer is about 15 to about 45 nucleotides long. In some embodiments, the second internal spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, or about 80 nucleotides long. In some embodiments, the first external spacer is about 10 to about 80 nucleotides long. In some embodiments, the first external spacer is about 15 to about 45 nucleotides long. In some embodiments, the first external spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, or about 80 nucleotides long. In some embodiments, the second external spacer is about 10 to about 80 nucleotides long. In some embodiments, the second external spacer is about 15 to about 45 nucleotides long. In some embodiments, the second external spacer is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, or about 80 nucleotides long.

In certain embodiments, the vectors, precursor RNA and circular RNA provided herein comprise an internal ribosome entry site (IRES) . Inclusion of an IRES permits the translation of one or more open reading frames from a circular RNA (e.g., open reading frames that form the expression sequence) . The IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19: 4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229: 295-298; Rees et al., BioTechniques (1996) 20: 102-110; Kobayashi et al., BioTechniques (1996) 21: 399-402; and Mosser et al., BioTechniques 1997 22 150-161) .

A multitude of IRES sequences are available and include sequences derived from a wide variety of viruses, such as from leader sequences of picornaviruses such as the encephalomyocarditis virus (EMCV) UTR (fang et al. J. Virol. (1989) 63: 1651-1660) , the polio leader sequence, the hepatitis A virus leader, the hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100 (25) : 15125-15130) , an IRES element from the foot and mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24: 2697-2700) , a giardiavirus IRES (Garlapati et al., J. Biol. Chem. (2004) 279 (5) : 3389-3397) , and the like.

In some embodiments, the polynucleotides herein comprise an expression sequence. In some embodiments, the expression sequence encodes a therapeutic protein.

In certain embodiments, the vectors provided herein comprise a 3′ UTR. In some embodiments, the 3′ UTR is from human beta globin, human alpha globin xenopus beta globin, xenopus alpha globin, human prolactin, human GAP-43, human eEFlal, human Tau, human TNFα, dengue virus, hantavirus small mRNA, bunyavirus small mRNA, turnip yellow mosaic virus, hepatitis C virus, rubella virus, tobacco mosaic virus, human IL-8, human actin, human GAPDH, human tubulin, hibiscus chlorotic ringspot virus, woodchuck hepatitis virus post translationally regulated element, sindbis virus, turnip crinkle virus, tobacco etch virus, or Venezuelan equine encephalitis virus.

In some embodiments, the vectors provided herein comprise a 5′ UTR. In some embodiments, the 5′ UTR is from human beta globin, Xenopus laevis beta globin, human alpha globin, Xenopus laevis alpha globin, rubella virus, tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein 70 kDa protein 1A, tobacco alcohol dehydrogenase, tobacco etch virus, turnip crinkle virus, or the adenovirus tripartite leader.

In some embodiments, a vector provided herein comprises a polyA region external of the 3′ and/or 5′ group I intron fragments. In some embodiments the polyA region is at least 15, 30, or 60 nucleotides long. In some embodiments, one or both polyA regions is 15-50 nucleotides long. In some embodiments, one or both polyA regions is 20-25 nucleotides long. The polyA sequence is removed upon circularization. Thus, an oligonucleotide hybridizing with the polyA sequence, such as a deoxythymine oligonucleotide (oligo (dT) ) conjugated to a solid surface (e.g., a resin) , can be used to separate circular RNA from its precursor RNA. Other sequences can also be disposed 5′ to the 3′ group I intron fragment or 3′ to the 5′ group I intron fragment and a complementary sequence can similarly be used for circular RNA purification.

In some embodiments, the DNA (e.g., vector) , linear RNA (e.g., precursor RNA) , and/or circular RNA polynucleotide provided herein is between 300 and 10000, 400 and 9000, 500 and 8000, 600 and 7000, 700 and 6000, 800 and 5000, 900 and 5000, 1000 and 5000, 1100 and 5000, 1200 and 5000, 1300 and 5000, 1400 and 5000, and/or 1500 and 5000 nucleotides in length. In some embodiments, the polynucleotide is at least 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, or 5000 nt in length. In some embodiments, the polynucleotide is no more than 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt in length. In some embodiments, the length of a DNA, linear RNA, and/or circular RNA polynucleotide provided herein is about 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt.

In some embodiments, provided herein is a vector. In certain embodiments, the vector comprises, in the following order, a) a first external complementary sequence, b) a 3′ group I intron fragment, c) optionally, a first spacer sequence, d) an IRES, e) an expression sequence, f) optionally, a second spacer sequence, g) a 5′ group I intron fragment, and h) a 3′ external complementary sequence. In some embodiments, the vector comprises a transcriptional promoter upstream of the 5′ homology region. In certain embodiments, the precursor RNA comprises, in the following order, a) a polyA sequence, b) an external spacer, c) a 3′ group I intron fragment, d) a first internal complementary sequence, e) an internal spacer, f) an IRES, g) an expression sequence, h) a stop codon cassette, i) optionally, an internal spacer, j) a second internal complementary sequence capable of forming a duplex with the first internal complementary sequence of d, k) a 5′ group I intron fragment, 1) an external spacer, and m) a polyA sequence.

In some embodiments, the circular RNA provided herein has higher functional stability than mRNA comprising the same expression sequence. In some embodiments, the circular RNA provided herein has higher functional stability than mRNA comprising the same expression sequence, 5moU modifications, an optimized UTR, a cap, and/or a polyA tail.

In some embodiments, the circular RNA polynucleotide provided herein has a functional half-life of at least 5 hours, 10 hours, 15 hours, 20 hours. 30 hours, 40 hours, 50 hours, 60 hours, 70 hours or 80 hours. In some embodiments, the circular RNA polynucleotide provided herein has a functional half-life of 5-80, 10-70, 15-60, and/or 20-50 hours. In some embodiments, the circular RNA polynucleotide provided herein has a functional half-life greater than (e.g., at least 1.5-fold greater than, at least 2-fold greater than) that of an equivalent linear RNA polynucleotide encoding the same protein. In some embodiments, functional half-life can be assessed through the detection of functional protein synthesis.

In some embodiments, the circular RNA polynucleotide provided herein has a half-life of at least 5 hours, 10 hours, 15 hours, 20 hours. 30 hours, 40 hours, 50 hours, 60 hours, 70 hours or 80 hours. In some embodiments, the circular RNA polynucleotide provided herein has a half-life of 5-80, 10-70, 15-60, and/or 20-50 hours. In some embodiments, the circular RNA polynucleotide provided herein has a half-life greater than (e.g., at least 1.5-fold greater than, at least 2-fold greater than) that of an equivalent linear RNA polynucleotide encoding the same protein. In some embodiments, the circular RNA polynucleotide, or pharmaceutical composition thereof, has a functional half-life in a human cell greater than or equal to that of a pre-determined threshold value. In some embodiments the functional half-life is determined by a functional protein assay. For example, in some embodiments, the functional half-life is determined by an in vitro luciferase assay, wherein the activity of Gaussia luciferase (GLuc) is measured in the media of human cells (e.g. HepG2) expressing the circular RNA polynucleotide every 1, 2, 6, 12, or 24 hours over 1, 2, 3, 4, 5, 6, 7, or 14 days. In other embodiments, the functional half-life is determined by an in vivo assay, wherein levels of a protein encoded by the expression sequence of the circular RNA polynucleotide are measured in patient serum or tissue samples every 1, 2, 6, 12, or 24 hours over 1, 2, 3, 4, 5, 6, 7, or 14 days. In some embodiments, the pre-determined threshold value is the functional half-life of a reference linear RNA polynucleotide comprising the same expression sequence as the circular RNA polynucleotide.

In some embodiments, the circular RNA provided herein may have a higher magnitude of expression than equivalent linear mRNA, e.g., a higher magnitude of expression 24 hours after administration of RNA to cells. In some embodiments, the circular RNA provided herein has a higher magnitude of expression than mRNA comprising the same expression sequence, 5moU modifications, an optimized UTR, a cap, and/or a polyA tail.

In some embodiments, the circular RNA provided herein may be less immunogenic than an equivalent mRNA when exposed to an immune system of an organism or a certain type of immune cell. In some embodiments, the circular RNA provided herein is associated with modulated production of cytokines when exposed to an immune system of an organism or a certain type of immune cell. For example, in some embodiments, the circular RNA provided herein is associated with reduced production of IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and/or TNFα when exposed to an immune system of an organism or a certain type of immune cell as compared to mRNA comprising the same expression sequence. In some embodiments, the circular RNA provided herein is associated with less IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and/or TNFα transcript induction when exposed to an immune system of an organism or a certain type of immune cell as compared to mRNA comprising the same expression sequence. In some embodiments, the circular RNA provided herein is less immunogenic than mRNA comprising the same expression sequence. In some embodiments, the circular RNA provided herein is less immunogenic than mRNA comprising the same expression sequence, 5moU modifications, an optimized UTR, a cap, and/or a polyA tail.

In certain embodiments, the circular RNA provided herein can be transfected into a cell as is, or can be transfected in DNA vector form and transcribed in the cell. Transcription of circular RNA from a transfected DNA vector can be via added polymerases or poylmerases encoded by nucleic acids transfected into the cell, or preferably via endogenous polymerases.

In certain embodiments, a circular RNA polynucleotide provided herein comprises modified RNA nucleotides and/or modified nucleosides.

In some embodiments, a linear precursor RNA or circular RNA molecule comprises a sequence set forth in Table 10 below.

Table 10 Exemplary linear precursor and circular RNA sequences.

5.3.4 Linear Nucleic Acid Molecules

In one aspect, the functional nucleic acid molecules provided herein are, or are configured to produce, a linear nucleic acid molecule, including a single-stranded linear RNA molecule. Accordingly, provided herein are linear RNAs (e.g., linear mRNAs) , and vectors (e.g., DNA vectors) that can be transcribed into the linear RNAs. The linear nucleic acid molecules described in this section can comprises any coding sequence as described in Section 5.3.1 (Coding Region) . The linear nucleic acid molecules descried in this section can comprise any untranslated region as described in Section 5.3.2 (Untranslated Regions (UTRs) ) .

In specific embodiments, the nucleic acid molecule of the present disclose comprises a 5’-UTR selected from SEQ ID NOS: 52 and 54. In specific embodiments, the nucleic acid molecule of the present disclose comprises a 5’-UTR selected from SEQ ID NOS: 53 and 55. In specific embodiments, the nucleic acid molecule of the present disclose comprises a 3’-UTR selected from SEQ ID NOS: 56 and 58. In specific embodiments, the nucleic acid molecule of the present disclose comprises a 3’-UTR selected from SEQ ID NOS: 57 and 59. In specific embodiments, the nucleic acid molecule of the present disclose comprises a 5’-UTR selected from SEQ ID NOS: 52 and 54, and a 3’-UTR selected from SEQ ID NOS: 56 and 58. In specific embodiments, the nucleic acid molecule of the present disclose comprises a 5’-UTR selected from SEQ ID NOS: 53 and 55, and a 3’-UTR selected from SEQ ID NOS: 57 and 59. In any of the embodiments described in this paragraph, the nucleic acid molecule may further comprise a coding region having a sequence as described in Section 5.3.1, such as any of the DNA coding sequences in Tables 2 to 4 or transcribed RNA sequences thereof. In particular embodiments, the nucleic acid molecules described in this paragraph can be RNA molecules transcribed in vitro. In some embodiments, a linear mRNA molecule comprises a sequence set forth in Table 11 below.

Table 11 Exemplary linear mRNA sequences.

In some embodiments, a linear nucleic acid sequence has a 5’ end and a 3’ end. In some embodiments, the linear nucleic acid molecules described herein comprises a 5’-cap structure as described herein. In some embodiments, the linear nucleic acid molecules described herein comprises a Poly-A region as described herein.

5.3.4.1 5’-Cap Structure

Without being bound by the theory, it is contemplated that, a 5’-cap structure of a polynucleotide is involved in nuclear export and increasing polynucleotide stability and binds the mRNA Cap Binding Protein (CBP) , which is responsible for polynucleotide stability in the cell and translation competency through the association of CBP with poly-A binding protein to form the mature cyclic mRNA species. The 5’-cap structure further assists the removal of 5’-proximal introns removal during mRNA splicing. Accordingly, in some embodiments, the nucleic acid molecules of the present disclosure comprise a 5’-cap structure.

Nucleic acid molecules may be 5’-end capped by the endogenous transcription machinery of a cell to generate a 5’-ppp-5’-triphosphate linkage between a terminal guanosine cap residue and the 5’-terminal transcribed sense nucleotide of the polynucleotide. This 5’-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5’ end of the polynucleotide may optionally also be 2’-O-methylated. 5’-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.

In some embodiments, the nucleic acid molecules of the present disclosure comprise one or more alterations to the natural 5’-cap structure generated by the endogenous process. Without being bound by the theory, a modification on the 5’-cap may increase the stability of polynucleotide, increase the half-life of the polynucleotide, and could increase the polynucleotide translational efficiency.

Exemplary alterations to the natural 5’-Cap structure include generation of a non-hydrolyzable cap structure preventing decapping and thus increasing polynucleotide half-life. In some embodiments, because cap structure hydrolysis requires cleavage of 5’-ppp-5’ phosphorodiester linkages, in some embodiments, modified nucleotides may be used during the capping reaction. For example, in some embodiments, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass. ) may be used with α-thio-guanosine nucleotides according to the manufacturer’s instructions to create a phosphorothioate linkage in the 5’-ppp-5’ cap. Additional modified guanosine nucleotides may be used, such as α-methyl-phosphonate and seleno-phosphate nucleotides.

Additional exemplary alterations to the natural 5’-Cap structure also include modification at the 2’-and/or 3’-position of a capped guanosine triphosphate (GTP) , a replacement of the sugar ring oxygen (that produced the carbocyclic ring) with a methylene moiety (CH₂) , a modification at the triphosphate bridge moiety of the cap structure, or a modification at the nucleobase (G) moiety.

Additional exemplary alterations to the natural 5’-cap structure include, but are not limited to, 2’-O-methylation of the ribose sugars of 5’-terminal and/or 5’-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2’-hydroxy group of the sugar. Multiple distinct 5’-cap structures can be used to generate the 5’-cap of a polynucleotide, such as an mRNA molecule. Additional exemplary 5’-Cap structures that can be used in connection with the present disclosure further include those described in International Patent Publication Nos. WO2008127688, WO 2008016473, and WO 2011015347, the entire contents of each of which are incorporated herein by reference.

In various embodiments, 5’-terminal caps can include cap analogs. Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type, or physiological) 5’-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and/linked to a polynucleotide.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanosines linked by a 5’-5’-triphosphate group, wherein one guanosine contains an N7-methyl group as well as a 3’-O-methyl group (i.e., N7, 3’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine, m⁷G-3’ mppp-G, which may equivalently be designated 3’ O-Me-m7G (5’) ppp (5’) G) . The 3’-O atom of the other, unaltered, guanosine becomes linked to the 5’-terminal nucleotide of the capped polynucleotide (e.g., an mRNA) . The N7-and 3’-O-methlyated guanosine provides the terminal moiety of the capped polynucleotide (e.g., mRNA) . Another exemplary cap structure is mCAP, which is similar to ARCA but has a 2’-O-methyl group on guanosine (i.e., N7, 2’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine, m⁷Gm-ppp-G) .

In some embodiments, a cap analog can be a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog may be modified at different phosphate positions with a boranophosphate group or a phophoroselenoate group such as the dinucleotide cap analogs described in U.S. Patent No.: 8,519,110, the entire content of which is herein incorporated by reference in its entirety.

In some embodiments, a cap analog can be a N7- (4-chlorophenoxyethyl) substituted dinucleotide cap analog known in the art and/or described herein. Non-limiting examples of N7- (4-chlorophenoxyethyl) substituted dinucleotide cap analogs include a N7- (4-chlorophenoxyethyl) -G (5’) ppp (5’) G and a N7- (4-chlorophenoxyethyl) -m3’-OG (5’) ppp (5’) G cap analog (see, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al. Bioorganic &Medicinal Chemistry 2013 21: 4570-4574; the entire content of which is herein incorporated by reference) . In other embodiments, a cap analog useful in connection with the nucleic acid molecules of the present disclosure is a 4-chloro/bromophenoxyethyl analog.

In various embodiments, a cap analog can include a guanosine analog. Useful guanosine analogs include but are not limited to inosine, N1-methyl-guanosine, 2’-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

Without being bound by the theory, it is contemplated that while cap analogs allow for the concomitant capping of a polynucleotide in an in vitro transcription reaction, up to 20%of transcripts remain uncapped. This, as well as the structural differences of a cap analog from the natural 5’-cap structures of polynucleotides produced by the endogenous transcription machinery of a cell, may lead to reduced translational competency and reduced cellular stability.

Accordingly, in some embodiments, a nucleic acid molecule of the present disclosure can also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5’-cap structures. As used herein, the phrase “more authentic” refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a “more authentic” feature is better representative of an endogenous, wild-type, natural or physiological cellular function, and/or structure as compared to synthetic features or analogs of the prior art, or which outperforms the corresponding endogenous, wild-type, natural, or physiological feature in one or more respects. Non-limiting examples of more authentic 5’-cap structures useful in connection with the nucleic acid molecules of the present disclosure are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5’-endonucleases, and/or reduced 5’-decapping, as compared to synthetic 5’-cap structures known in the art (or to a wild-type, natural or physiological 5’-cap structure) . For example, in some embodiments, recombinant Vaccinia Virus Capping Enzyme and recombinant 2’-O-methyltransferase enzyme can create a canonical 5’-5’-triphosphate linkage between the 5’-terminal nucleotide of a polynucleotide and a guanosine cap nucleotide wherein the cap guanosine contains an N7-methylation and the 5’-terminal nucleotide of the polynucleotide contains a 2’-O-methyl. Such a structure is termed the Cap1 structure. This cap results in a higher translational-competency, cellular stability, and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5’ cap analog structures known in the art. Other exemplary cap structures include 7mG (5’) ppp (5’) N, pN2p (Cap 0) , 7mG (5’) ppp (5’) NlmpNp (Cap 1) , 7mG (5’) -ppp (5’) NlmpN2mp (Cap 2) , and m (7) Gpppm (3) (6, 6, 2’) Apm (2’) Apm (2’) Cpm (2) (3, 2’) Up (Cap 4) .

Without being bound by the theory, it is contemplated that the nucleic acid molecules of the present disclosure can be capped post-transcriptionally, and because this process is more efficient, nearly 100%of the nucleic acid molecules may be capped.

5.3.4.2 The Polyadenylation (Poly-A) Regions

During natural RNA processing, a long chain of adenosine nucleotides (poly-A region) is normally added to messenger RNA (mRNA) molecules to increase the stability of the molecule. Immediately after transcription, the 3’-end of the transcript is cleaved to free a 3’-hydroxy. Then poly-A polymerase adds a chain of adenosine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A region that is between 100 and 250 residues long. Without being bound by the theory, it is contemplated that a poly-A region can confer various advantages to the nucleic acid molecule of the present disclosure.

Accordingly, in some embodiments, a nucleic acid molecule of the present disclosure (e.g., an mRNA) comprises a polyadenylation signal. In some embodiments, a nucleic acid molecule of the present disclosure (e.g., an mRNA) comprises one or more polyadenylation (poly-A) regions. In some embodiments, a poly-A region is composed entirely of adenine nucleotides or functional analogs thereof. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 3’-end. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 5’-end. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 5’-end and at least one poly-A region at its 3’-end.

According to the present disclosure, the poly-A region can have varied lengths in different embodiments. Particularly, in some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 30 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 35 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 40 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 45 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 50 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 55 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 60 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 65 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 70 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 75 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 80 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 85 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 90 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 95 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 100 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 110 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 120 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 130 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 140 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 150 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 160 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 170 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 180 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 190 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 200 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 225 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 250 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 275 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 300 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 350 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 400 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 450 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 500 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 600 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 700 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 800 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 900 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1000 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1100 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1200 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1300 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1400 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1500 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1600 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1700 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1800 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1900 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 2000 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 2250 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 2500 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 2750 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 3000 nucleotides in length.

In some embodiments, length of a poly-A region in a nucleic acid molecule can be selected based on the overall length of the nucleic acid molecule, or a portion thereof (such as the length of the coding region or the length of an open reading frame of the nucleic acid molecule, etc. ) . For example, in some embodiments, the poly-A region accounts for about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or more of the total length of nucleic acid molecule containing the poly-A region.

Without being bound by the theory, it is contemplated that certain RNA-binding proteins can bind to the poly-A region located at the 3’-end of an mRNA molecule. These poly-A binding proteins (PABP) can modulate mRNA expression, such as interacting with translation initiation machinery in a cell and/or protecting the 3’-poly-A tails from degradation. Accordingly, in some embodiments, the nucleic acid molecule of the present disclosure (e.g., mRNA) comprises at least one binding site for poly-A binding protein (PABP) . In other embodiments, the nucleic acid molecule is conjugated or complex with a PABP before loaded into a delivery vehicle (e.g., lipid nanoparticles) .

In some embodiments, the nucleic acid molecule of the present disclosure (e.g., mRNA) comprises a poly-A-G Quartet. The G-quartet is a cyclic hydrogen bonded array of four guanosine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A region. The resultant polynucleotides (e.g., mRNA) may be assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the polyA-G quartet structure results in protein production equivalent to at least 75%of that seen using a poly-A region of 120 nucleotides alone.

In some embodiments, the nucleic acid molecule of the present disclosure (e.g., mRNA) may include a poly-A region and may be stabilized by the addition of a 3’-stabilizing region. In some embodiments, the 3’-stabilizing region which may be used to stabilize a nucleic acid molecule (e.g., mRNA) including the poly-A or poly-A-G Quartet structures as described in International Patent Publication No. WO2013/103659, the content of which is incorporated herein by reference in its entirety.

In other embodiments, the 3’-stabilizing region which may be used in connection with the nucleic acid molecules of the present disclosure include a chain termination nucleoside such as but is not limited to 3’-deoxyadenosine (cordycepin) , 3’-deoxyuridine, 3’-deoxycytosine, 3’-deoxyguanosine, 3’-deoxythymine, 2’, 3’-dideoxynucleosides, such as 2’, 3’-dideoxyadenosine, 2’, 3’-dideoxyuridine, 2’, 3’-dideoxycytosine, 2’, 3’-dideoxyguanosine, 2’, 3’-dideoxythymine, a 2’-deoxynucleoside, or an O-methylnucleoside, 3’-deoxynucleoside, 2’, 3’-dideoxynucleoside 3’-O-methylnucleosides, 3’-O-ethylnucleosides, 3’-arabinosides, and other alternative nucleosides known in the art and/or described herein.

5.3.5 Secondary Structure

Without being bound by the theory, it is contemplated that a stem-loop structure can direct RNA folding, protect structural stability of a nucleic acid molecule (e.g., mRNA) , provide recognition sites for RNA binding proteins, and serve as a substrate for enzymatic reactions. For example, the incorporation of a miR sequence and/or a TEE sequence changes the shape of the stem loop region which may increase and/or decrease translation (Kedde et al. A Pumilio-induced RNA structure switch in p27- 3’UTR controls miR-221 and miR-222 accessibility. Nat Cell Biol., 2010 Oct; 12 (10) : 1014-20, the content of which is herein incorporated by reference in its entirety) .

Accordingly, in some embodiments, the nucleic acid molecules as described herein (e.g., mRNA) or a portion thereof may assume a stem-loop structure, such as but is not limited to a histone stem loop. In some embodiments, the stem-loop structure is formed from a stem-loop sequence that is about 25 or about 26 nucleotides in length such as, but not limited to, those as described in International Patent Publication No. WO2013/103659, the content of which is incorporated herein by reference in its entirety. Additional examples of stem-loop sequences include those described in International Patent Publication No. WO2012/019780 and International Patent Publication No. WO201502667, the contents of which are incorporated herein by reference. In some embodiments, the step-loop sequence comprises a TEE as described herein. In some embodiments, the step-loop sequence comprises a miR sequence as described herein. In specific embodiments, the stem loop sequence may include a miR-122 seed sequence.

In some embodiments, the nucleic acid molecule of the present disclosure (e.g., mRNA) comprises a stem-loop sequence located upstream (to the 5’-end) of the coding region in a nucleic acid molecule. In some embodiments, the stem-loop sequence is located within the 5’-UTR of the nucleic acid molecule. In some embodiments, the nucleic acid molecule of the present disclosure (e.g., mRNA) comprises a stem-loop sequence located downstream (to the 3’-end) of the coding region in a nucleic acid molecule. In some embodiments, the stem-loop sequence is located within the 3’-UTR of the nucleic acid molecule. In some cases, a nucleic acid molecule can contain more than one stem-loop sequences. In some embodiment, the nucleic acid molecule comprises at least one stem-loop sequence in the 5’-UTR, and at least one stem-loop sequence in the 3’-UTR.

In some embodiments, a nucleic acid molecule comprising a stem-loop structure further comprises a stabilization region. In some embodiment, the stabilization region comprises at least one chain terminating nucleoside that functions to slow down degradation and thus increases the half-life of the nucleic acid molecule. Exemplary chain terminating nucleoside that can be used in connection with the present disclosure include but are not limited to 3’-deoxyadenosine (cordycepin) , 3’-deoxyuridine, 3’-deoxycytosine, 3’-deoxyguanosine, 3’-deoxythymine, 2’, 3’-dideoxynucleosides, such as 2’, 3’-dideoxyadenosine, 2’, 3’-dideoxyuridine, 2’, 3’-dideoxycytosine, 2’, 3’-dideoxyguanosine, 2’, 3’-dideoxythymine, a 2’-deoxynucleoside, or an O-methylnucleoside, 3’-deoxynucleoside, 2’, 3’-dideoxynucleoside 3’-O-methylnucleosides, 3’-O-ethylnucleosides, 3’-arabinosides, and other alternative nucleosides known in the art and/or described herein. In other embodiments, a stem-loop structure may be stabilized by an alteration to the 3’-region of the polynucleotide that can prevent and/or inhibit the addition of oligio (U) (International Patent Publication No. WO2013/103659, incorporated herein by reference in its entirety) .

In some embodiments, a nucleic acid molecule of the present disclosure comprises at least one stem-loop sequence and a poly-A region or polyadenylation signal. Non-limiting examples of polynucleotide sequences comprising at least one stem-loop sequence and a poly-A region or a polyadenylation signal include those described in International Patent Publication No. WO2013/120497, International Patent Publication No. WO2013/120629, International Patent Publication No. WO2013/120500, International Patent Publication No. WO2013/120627, International Patent Publication No. WO2013/120498, International Patent Publication No. WO2013/120626, International Patent Publication No. WO2013/120499 and International Patent Publication No. WO2013/120628, the content of each of which is incorporated herein by reference in its entirety.

In some embodiments, the nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal can encode for a pathogen antigen or fragment thereof such as the polynucleotide sequences described in International Patent Publication No. WO2013/120499 and International Patent Publication No. WO2013/120628, the content of each of which is incorporated herein by reference in its entirety.

In some embodiments, the nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal can encode for a therapeutic protein such as the polynucleotide sequences described in International Patent Publication No. WO2013/120497 and International Patent Publication No. WO2013/120629, the content of each of which is incorporated herein by reference in its entirety.

In some embodiments, the nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal can encode for a tumor antigen or fragment thereof such as the polynucleotide sequences described in International Patent Publication No. WO2013/120500 and International Patent Publication No. WO2013/120627, the content of each of which is incorporated herein by reference in its entirety.

In some embodiments, the nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal can code for an allergenic antigen or an autoimmune self-antigen such as the polynucleotide sequences described in International Patent Publication No. WO2013/120498 and International Patent Publication No. WO2013/120626, the content of each of which is incorporated herein by reference in its entirety.

5.3.6 Functional nucleotide analogs

In some embodiments, a payload nucleic acid molecule described herein contains only canonical nucleotides selected from A (adenosine) , G (guanosine) , C (cytosine) , U (uridine) , and T (thymidine) . Without being bound by the theory, it is contemplated that certain functional nucleotide analogs can confer useful properties to a nucleic acid molecule. Examples of such as useful properties in the context of the present disclosure include but are not limited to increased stability of the nucleic acid molecule, reduced immunogenicity of the nucleic acid molecule in inducing innate immune responses, enhanced production of protein encoded by the nucleic acid molecule, increased intracellular delivery and/or retention of the nucleic acid molecule, and/or reduced cellular toxicity of the nucleic acid molecule, etc.

Accordingly, in some embodiments, a payload nucleic acid molecule comprises at least one functional nucleotide analog as described herein. In some embodiments, the functional nucleotide analog contains at least one chemical modification to the nucleobase, the sugar group and/or the phosphate group. Accordingly, a payload nucleic acid molecule comprising at least one functional nucleotide analog contains at least one chemical modification to the nucleobases, the sugar groups, and/or the internucleoside linkage. Exemplary chemical modifications to the nucleobases, sugar groups, or internucleoside linkages of a nucleic acid molecule are provided herein.

As described herein, ranging from 0%to 100%of all nucleotides in a payload nucleic acid molecule can be functional nucleotide analogs as described herein. For example, in various embodiments, from about 1%to about 20%, from about 1%to about 25%, from about 1%to about 50%, from about 1%to about 60%, from about 1%to about 70%, from about 1%to about 80%, from about 1%to about 90%, from about 1%to about 95%, from about 10%to about 20%, from about 10%to about 25%, from about 10%to about 50%, from about 10%to about 60%, from about 10%to about 70%, from about 10%to about 80%, from about 10%to about 90%, from about 10%to about 95%, from about 10%to about 100%, from about 20%to about 25%, from about 20%to about 50%, from about 20%to about 60%, from about 20%to about 70%, from about 20%to about 80%, from about 20%to about 90%, from about 20%to about 95%, from about 20%to about 100%, from about 50%to about 60%, from about 50%to about 70%, from about 50%to about 80%, from about 50%to about 90%, from about 50%to about 95%, from about 50%to about 100%, from about 70%to about 80%, from about 70%to about 90%, from about 70%to about 95%, from about 70%to about 100%, from about 80%to about 90%, from about 80%to about 95%, from about 80%to about 100%, from about 90%to about 95%, from about 90%to about 100%, or from about 95%to about 100%of all nucleotides in a nucleic acid molecule are functional nucleotide analogs described herein. In any of these embodiments, a functional nucleotide analog can be present at any position (s) of a nucleic acid molecule, including the 5’-terminus, 3’-terminus, and/or one or more internal positions. In some embodiments, a single nucleic acid molecule can contain different sugar modifications, different nucleobase modifications, and/or different types internucleoside linkages (e.g., backbone structures) .

As described herein, ranging from 0%to 100%of all nucleotides of a kind (e.g., all purine-containing nucleotides as a kind, or all pyrimidine-containing nucleotides as a kind, or all A, G, C, T or U as a kind) in a payload nucleic acid molecule can be functional nucleotide analogs as described herein. For example, in various embodiments, from about 1%to about 20%, from about 1%to about 25%, from about 1%to about 50%, from about 1%to about 60%, from about 1%to about 70%, from about 1%to about 80%, from about 1%to about 90%, from about 1%to about 95%, from about 10%to about 20%, from about 10%to about 25%, from about 10%to about 50%, from about 10%to about 60%, from about 10%to about 70%, from about 10%to about 80%, from about 10%to about 90%, from about 10%to about 95%, from about 10%to about 100%, from about 20%to about 25%, from about 20%to about 50%, from about 20%to about 60%, from about 20%to about 70%, from about 20%to about 80%, from about 20%to about 90%, from about 20%to about 95%, from about 20%to about 100%, from about 50%to about 60%, from about 50%to about 70%, from about 50%to about 80%, from about 50%to about 90%, from about 50%to about 95%, from about 50%to about 100%, from about 70%to about 80%, from about 70%to about 90%, from about 70%to about 95%, from about 70%to about 100%, from about 80%to about 90%, from about 80%to about 95%, from about 80%to about 100%, from about 90%to about 95%, from about 90%to about 100%, or from about 95%to about 100%of a kind of nucleotides in a nucleic acid molecule are functional nucleotide analogs described herein. In any of these embodiments, a functional nucleotide analog can be present at any position (s) of a nucleic acid molecule, including the 5’-terminus, 3’-terminus, and/or one or more internal positions. In some embodiments, a single nucleic acid molecule can contain different sugar modifications, different nucleobase modifications, and/or different types internucleoside linkages (e.g., backbone structures) .

5.3.7 Modification to Nucleobases

In some embodiments, a functional nucleotide analog contains a non-canonical nucleobase. In some embodiments, canonical nucleobases (e.g., adenine, guanine, uracil, thymine, and cytosine) in a nucleotide can be modified or replaced to provide one or more functional analogs of the nucleotide. Exemplary modification to nucleobases include but are not limited to one or more substitutions or modifications including but not limited to alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and/or thio substitutions; one or more fused or open rings, oxidation, and/or reduction.

In some embodiments, the non-canonical nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having an modified uracil include pseudouridine (ψ) , pyridin-4-one ribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s²U) , 4-thio-uracil (s⁴U) , 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho⁵U) , 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil) , 3-methyl-uracil (m³U) , 5-methoxy-uracil (mo⁵U) , uracil 5-oxyacetic acid (cmo⁵U) , uracil 5-oxyacetic acid methyl ester (mcmo⁵U) , 5-carboxymethyl-uracil (cm⁵U) , 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm⁵U) , 5-carboxyhydroxymethyl-uracil methyl ester (mchm⁵U) , 5-methoxycarbonylmethyl-uracil (mcm⁵U) , 5-methoxycarbonylmethyl-2-thio-uracil (mcm⁵s²U) , 5-aminomethyl-2-thio-uracil (nm⁵s²U) , 5-methylaminomethyl-uracil (mnm⁵U) , 5-methylaminomethyl-2-thio-uracil (mnm⁵s²U) , 5-methylaminomethyl-2-seleno-uracil (mnm⁵se²U) , 5-carbamoylmethyl-uracil (ncm⁵U) , 5-carboxymethylaminomethyl-uracil (cmnm⁵U) , 5-carboxymethylaminomethyl-2-thio-uracil (cmnm⁵s²U) , 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-taurinomethyl-uracil (τm ⁵U) , 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uracil (τm⁵5s²U) , 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uracil (m⁵U, i.e., having the nucleobase deoxythymine) , 1-methyl-pseudouridine (m¹ψ) , 1-ethyl-pseudouridine (Et¹ψ) , 5-methyl-2-thio-uracil (m⁵s²U) , 1-methyl-4-thio-pseudouridine (m¹s⁴ψ) , 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ) , 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D) , dihydropseudouridine, 5, 6-dihydrouracil, 5-methyl-dihydrouracil (m⁵D) , 2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3- (3-amino-3-carboxypropyl) uracil (acp³U) , 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine (acp³ψ) , 5- (isopentenylaminomethyl) uracil (m⁵U) , 5- (isopentenylaminomethyl) -2-thio-uracil (m⁵s²U) , 5, 2’-O-dimethyl-uridine (m⁵Um) , 2-thio-2’-O-methyl-uridine (s²Um) , 5-methoxycarbonylmethyl-2’-O-methyl-uridine (mcm⁵Um) , 5-carbamoylmethyl-2’-O-methyl-uridine (ncm⁵Um) , 5-carboxymethylaminomethyl-2’-O-methyl-uridine (cmnm⁵Um) , 3, 2’-O-dimethyl-uridine (m³Um) , and 5- (isopentenylaminomethyl) -2’-O-methyl-uridine (inm⁵Um) , 1-thio-uracil, deoxythymidine, 5- (2-carbomethoxyvinyl) -uracil, 5- (carbamoylhydroxymethyl) -uracil, 5-carbamoylmethyl-2-thio-uracil, 5-carboxymethyl-2-thio-uracil, 5-cyanomethyl-uracil, 5-methoxy-2-thio-uracil, and 5- [3- (1-E-propenylamino) ] uracil.

In some embodiments, the non-canonical nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytosine, 6-aza-cytosine, pseudoisocytidine, 3-methyl-cytosine (m3C) , N4-acetyl-cytosine (ac4C) , 5-formyl-cytosine (f5C) , N4-methyl-cytosine (m4C) , 5-methyl-cytosine (m5C) , 5-halo-cytosine (e.g., 5-iodo-cytosine) , 5-hydroxymethyl-cytosine (hm5C) , 1-methyl-pseudoisocytidine, pyrrolo-cytosine, pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C) , 2-thio-5-methyl-cytosine, 4-thio-pseudoisocytidine, 4-thio-1- methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytosine, 2-methoxy-5-methyl-cytosine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C) , 5, 2’-O-dimethyl-cytidine (m5Cm) , N4-acetyl-2’-O-methyl-cytidine (ac4Cm) , N4, 2’-O-dimethyl-cytidine (m4Cm) , 5-formyl-2’-O-methyl-cytidine (fSCm) , N4, N4, 2’-O-trimethyl-cytidine (m42Cm) , 1-thio-cytosine, 5-hydroxy-cytosine, 5- (3-azidopropyl) -cytosine, and 5- (2-azidoethyl) -cytosine.

In some embodiments, the non-canonical nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having an alternative adenine include 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine) , 6-halo-purine (e.g., 6-chloro-purine) , 2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyl-adenine (m1A) , 2-methyl-adenine (m2A) , N6-methyl-adenine (m6A) , 2-methylthio-N6-methyl-adenine (ms2m6A) , N6-isopentenyl-adenine (i6A) , 2-methylthio-N6-isopentenyl-adenine (ms2i6A) , N6- (cis-hydroxyisopentenyl) adenine (io6A) , 2-methylthio-N6- (cis-hydroxyisopentenyl) adenine (ms2io6A) , N6-glycinylcarbamoyl-adenine (g6A) , N6-threonylcarbamoyl-adenine (t6A) , N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A) , 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A) , N6, N6-dimethyl-adenine (m62A) , N6-hydroxynorvalylcarbamoyl-adenine (hn6A) , 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A) , N6-acetyl-adenine (ac6A) , 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, N6, 2’-O-dimethyl-adenosine (m6Am) , N6, N6, 2’-O-trimethyl-adenosine (m62Am) , 1, 2’-O-dimethyl-adenosine (m1Am) , 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, N6- (19-amino-pentaoxanonadecyl) -adenine, 2, 8-dimethyl-adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine.

In some embodiments, the non-canonical nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I) , 1-methyl-inosine (m1I) , wyosine (imG) , methylwyosine (mimG) , 4-demethyl-wyosine (imG-14) , isowyosine (imG2) , wybutosine (yW) , peroxywybutosine (o2yW) , hydroxywybutosine (OHyW) , undermodified hydroxywybutosine (OHyW*) , 7-deaza-guanine, queuosine (Q) , epoxyqueuosine (oQ) , galactosyl-queuosine (galQ) , mannosyl-queuosine (manQ) , 7-cyano-7-deaza-guanine (preQO) , 7-aminomethyl-7-deaza-guanine (preQ1) , archaeosine (G+) , 7-deaza-8-aza-guanine, 6-thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G) , 6-thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (m1G) , N2-methyl-guanine (m2G) , N2, N2-dimethyl-guanine (m22G) , N2, 7-dimethyl-guanine (m2, 7G) , N2, N2, 7-dimethyl-guanine (m2, 2, 7G) , 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2, N2-dimethyl-6-thio-guanine, N2- methyl-2’-O-methyl-guanosine (m2Gm) , N2, N2-dimethyl-2’-O-methyl-guanosine (m22Gm) , 1-methyl-2’-O-methyl-guanosine (m1Gm) , N2, 7-dimethyl-2’-O-methyl-guanosine (m2, 7Gm) , 2’-O-methyl-inosine (Im) , 1, 2’-O-dimethyl-inosine (m1Im) , 1-thio-guanine, and O-6-methyl-guanine.

In some embodiments, the non-canonical nucleobase of a functional nucleotide analog can be independently a purine, a pyrimidine, a purine or pyrimidine analog. For example, in some embodiments, the non-canonical nucleobase can be modified adenine, cytosine, guanine, uracil, or hypoxanthine. In other embodiments, the non-canonical nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo [3, 4-d] pyrimidines, 5-methylcytosine (5-me-C) , 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil) , 4-thiouracil, 8-halo (e.g., 8-bromo) , 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo [3, 4-d] pyrimidine, imidazo [1, 5-a] 1, 3, 5 triazinones, 9-deazapurines, imidazo [4, 5-d] pyrazines, thiazolo [4, 5-d] pyrimidines, pyrazin-2-ones, 1, 2, 4-triazine, pyridazine; or 1, 3, 5 triazine.

5.3.8 Modification to the Sugar

In some embodiments, a functional nucleotide analog contains a non-canonical sugar group. In various embodiments, the non-canonical sugar group can be a 5-carbon or 6-carbon sugar (such as pentose, ribose, arabinose, xylose, glucose, galactose, or a deoxy derivative thereof) with one or more substitutions, such as a halo group, a hydroxy group, a thiol group, an alkyl group, an alkoxy group, an alkenyloxy group, an alkynyloxy group, an cycloalkyl group, an aminoalkoxy group, an alkoxyalkoxy group, an hydroxyalkoxy group, an amino group, an azido group, an aryl group, an aminoalkyl group, an aminoalkenyl group, an aminoalkynyl group, etc.

Generally, RNA molecules contains the ribose sugar group, which is a 5-membered ring having an oxygen. Exemplary, non-limiting alternative nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene) ; addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl) ; ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane) ; ring expansion of ribose (e.g., to form a 6-or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino (that also has a phosphoramidate backbone) ) ; multicyclic forms (e.g., tricyclo and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds) , threose nucleic acid (TNA, where ribose is replace with α-L-threofuranosyl- (3’→2’) ) , and peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone) .

In some embodiments, the sugar group contains one or more carbons that possess the opposite stereochemical configuration of the corresponding carbon in ribose. Thus, a nucleic acid molecule can include nucleotides containing, e.g., arabinose or L-ribose, as the sugar. In some embodiments, the nucleic acid molecule includes at least one nucleoside wherein the sugar is L-ribose, 2’-O-methyl-ribose, 2’-fluoro-ribose, arabinose, hexitol, an LNA, or a PNA.

5.3.9 Modifications to the Internucleoside Linkage

In some embodiments, the payload nucleic acid molecule of the present disclosure can contain one or more modified internucleoside linkage (e.g., phosphate backbone) . Backbone phosphate groups can be altered by replacing one or more of the oxygen atoms with a different substituent.

In some embodiments, the functional nucleotide analogs can include the replacement of an unaltered phosphate moiety with another internucleoside linkage as described herein. Examples of alternative phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be altered by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates) , sulfur (bridged phosphorothioates) , and carbon (bridged methylene-phosphonates) .

The alternative nucleosides and nucleotides can include the replacement of one or more of the non-bridging oxygens with a borane moiety (BH₃) , sulfur (thio) , methyl, ethyl, and/or methoxy. As a non-limiting example, two non-bridging oxygens at the same position (e.g., the alpha (α) , beta (β) or gamma (γ) position) can be replaced with a sulfur (thio) and a methoxy. The replacement of one or more of the oxygen atoms at the position of the phosphate moiety (e.g., α-thio phosphate) is provided to confer stability (such as against exonucleases and endonucleases) to RNA and DNA through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.

Other internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein.

Additional examples of nucleic acid molecules (e.g., mRNA) , compositions, formulations and/or methods associated therewith that can be used in connection with the present disclosure further include those described in WO2002/098443, WO2003/051401, WO2008/052770, WO2009127230, WO2006122828, WO2008/083949, WO2010088927, WO2010/037539, WO2004/004743, WO2005/016376, WO2006/024518, WO2007/095976, WO2008/014979, WO2008/077592, WO2009/030481, WO2009/095226, WO2011069586, WO2011026641, WO2011/144358, WO2012019780, WO2012013326, WO2012089338, WO2012113513, WO2012116811, WO2012116810, WO2013113502, WO2013113501, WO2013113736, WO2013143698, WO2013143699, WO2013143700, WO2013/120626, WO2013120627, WO2013120628, WO2013120629, WO2013174409, WO2014127917, WO2015/024669, WO2015/024668, WO2015/024667, WO2015/024665, WO2015/024666, WO2015/024664, WO2015101415, WO2015101414, WO2015024667, WO2015062738, WO2015101416, the content of each of which is incorporated herein in its entirety.

Therapeutic nucleic acid molecules as described herein can be isolated or synthesized using methods known in the art. In some embodiments, DNA or RNA molecules to be used in connection with the present disclosure are chemically synthesized. In other embodiments, DNA or RNA molecules to be used in connection with the present disclosure are isolated from a natural source.

In some embodiments, mRNA molecules to be used in connection with the present disclosure are biosynthesized using a host cell. In particular embodiments, an mRNA is produced by transcribing a corresponding DNA sequencing using a host cell. In some embodiments, a DNA sequence encoding an mRNA sequence is incorporated into an expression vector, which vector is then introduced into a host cell (e.g., E. coli) using methods known in the art. The host cell is then cultured under a suitable condition to produce mRNA transcripts. Other methods for producing an mRNA molecule from an encoding DNA are known in the art. For example, in some embodiments, a cell-free (in vitro) transcription system comprising enzymes of the transcription machinery of a host cell can be used to produce mRNA transcripts. An exemplary cell-free transcription reaction system is described in Example 1 of the present disclosure.

5.4 Methods

As described herein, nucleic acid molecules (e.g., RNA molecules) encoding one or more reprogramming factors comprising a NeuroD1 polypeptide can be packaged into lipid nanoparticles and efficiently expressed by target glial cells upon delivery of the lipid nanoparticles to the glial cell. Such lipid nanoparticle compositions can be used to convert the glial cells into function neurons both in vitro and in vivo.

In one aspect, provided herein is a method for reprogramming glial cells into functional neurons. In some embodiments, the method comprises contacting a starting population of glial cells with lipid nanoparticles comprising nucleic acid molecules encoding one or more reprogramming factors comprising a NeuroD1 polypeptide. In some embodiments, upon the contacting, the glial cells express the one or more reprogramming factors encoded by the nucleic acid molecules. In some embodiments, the glial cells start to express the encoded one or more reprogramming factor within 24 hours, within 18 hours, within 12 hours or within 6 hours after the lipid nanoparticles are contacted with the glial cells. In some embodiments, the glial cells start to undergo trans-differentiation within less than 7 days, within less than 6 days, within less than 5 days, within less than 4 days, or within less than 3 days after the lipid nanoparticles are contacted with the glial cells. In some embodiments, the glial cells are converted into functional neurons within less than 21 days, less than 14 days, within less than 13 days, within less than 12 days, within less than 11 days, within less than 10 days, within less than 9 days, within less than 8 days, or within less than 7 days after the lipid nanoparticles are contacted with the glial cells.

The lipid nanoparticles used in the present method are described in Section 5.5 (Nanoparticle Compositions) , and the nucleic acid molecules encompassed in the lipid nanoparticles are described in Section 5.3 (Functional Nucleic Acids) . In some embodiments, the nucleic acids encompassed in the lipid nanoparticles can be one species of nucleic acid, or more than one species of different nucleic acids. In some embodiments, the nucleic acids in the lipid nanoparticles encode one or more reprogramming factors comprising a NeuroD1 polypeptide. In some embodiments, the nucleic acid comprises an expression sequence that is selected from any expression sequences or coding sequences as described in Section 5.3.1 (Coding Region) . In some embodiments, the nucleic acids in the lipid nanoparticles can be linear RNA molecules, such as mRNA molecules, as described in Section 5.3.4 (Linear Nucleic Acid Molecules) . In some embodiments, the nucleic acids in the lipid nanoparticles can be circular RNA molecules as described in Section 5.3.3 (Circular Nucleic Acid Molecules) . In specific embodiments, the encoded NeuroD1 polypeptide is a wild-type NeuroD1. In some embodiments, the encoded NeuroD1 is human NeuroD1 having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the encoded NeuroD1 is a NeuroD1 polypeptide having the amino acid sequence of SEQ ID NO: 2, where an extra V encoded by a Kozak fragment is located at the second residue. In alternative embodiments, the encoded NeuroD1 polypeptide is a functional derivative of NeuroD1. In some embodiments, a functional derivative of NeuroD1 shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) NeuroD1 protein from which it derives.

In some embodiments, the starting population of glial cells comprise astrocytes. In some embodiments, the astrocytes in the starting population express one or more glial cell markers selected from GFAP, Aldh1l1, S100β and Sox9. In some embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding a NeuroD1 polypeptide. In alternative embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding a NeuroD1 polypeptide and at least one second polypeptide selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof. In particular embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (a) a NeuroD1 polypeptide alone. In alternative embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (b) a NeuroD1 polypeptide and a Dlx2 polypeptide. In alternative embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (c) a NeuroD1 polypeptide and a Isl1 polypeptide. In alternative embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (d) a NeuroD1 polypeptide and a Ascl1 polypeptide. In alternative embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (e) a NeuroD1 polypeptide and a Ngn2 polypeptide. In alternative embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (f) a NeuroD1 polypeptide, a Ascl1 polypeptide, a LMX1A polypeptide and a microRNA miR218. In alternative embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (g) a NeuroD1 polypeptide, a Ascl1 polypeptide, and a Dlx2 polypeptide. In alternative embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (h) a NeuroD1 polypeptide and a Ctip2 polypeptide. In alternative embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (i) a NeuroD1 polypeptide, a Ascl1 polypeptide, a Dlx2 polypeptide, and a Ctip2 polypeptide. In alternative embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (j) a NeuroD1 polypeptide, a Ascl1 polypeptide, aDlx2 polypeptide, and a Isl1 polypeptide. In alternative embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (k) a NeuroD1 polypeptide, a Ascl1 polypeptide, and a Isl1 polypeptide. In alternative embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (l) a NeuroD1 polypeptide, a Ascl1 polypeptide, and a Ctip2 polypeptide. In alternative embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (m) a NeuroD1 polypeptide, a Dlx2 polypeptide, and a Ctip2 polypeptide. In alternative embodiments, the astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (n) a NeuroD1 polypeptide, a Dlx2 polypeptide, and a Ngn2 polypeptide.

In some embodiments, upon contacting the population of astrocytes with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the astrocytes stop expressing one or more glial markers selected from GFAP, Aldh1l1, S100β and Sox9. In some embodiments, the astrocytes stop expressing the one or more glial markers within less than 7 days, less than 6 days, less than 5 days, less than 4 days, or less than 3 days after contacting with the lipid nanoparticles.

In some embodiments, upon contacting the population of astrocytes with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the astrocytes exhibit one or more neuronal phenotypes. In some embodiments, the one or more neuronal phenotypes comprise expression of one or more neuronal markers selected from DCX, TUJ1, NeuN, and MAP2. In some embodiments, the one or more neuronal phenotypes comprise ability of firing action potentials. In some embodiments, the one or more neuronal phenotypes comprise formation of dendrites and/or exons on the cell surface. In some embodiments, the one or more neuronal phenotypes comprise formation of synapses with a neighboring cell. In some embodiments, the one or more neuronal phenotypes comprise the ability of releasing synaptic currents. In some embodiments, the synaptic currents are glutamatergic current, GABAergic current, Dopaminergic current, glycinergic current, serotonergic current or norepinephrinergic current.

In some embodiments, upon contacting the population of astrocytes with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the astrocytes are converted into functional neurons. In some embodiments, the functional neurons are selected from glutamatergic neurons, GABAergic neurons, dopaminergic neurons; motor neurons, glycinergic neurons, serotonergic neurons, norepinephrinergic neurons, and sensory neurons. In some embodiments, the astrocytes are converted into functional neurons less than 21 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, or less than 7 days after contacting with the lipid nanoparticles.

In some embodiments, the starting population of glial cells comprise NG-2 cells. In some embodiments, the NG-2 cells in the starting population express one or more glial cell markers selected from GFAP, Aldh1l1, S100β and Sox9. In some embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding a NeuroD1 polypeptide. In alternative embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding a NeuroD1 polypeptide and at least one second polypeptide selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof. In particular embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (a) a NeuroD1 polypeptide alone. In alternative embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (b) a NeuroD1 polypeptide and a Dlx2 polypeptide. In alternative embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (c) a NeuroD1 polypeptide and a Isl1 polypeptide. In alternative embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (d) a NeuroD1 polypeptide and a Ascl1 polypeptide. In alternative embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (e) a NeuroD1 polypeptide and a Ngn2 polypeptide. In alternative embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (f) a NeuroD1 polypeptide, a Ascl1 polypeptide, a LMX1A polypeptide and a microRNA miR218. In alternative embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (g) a NeuroD1 polypeptide, a Ascl1 polypeptide, and a Dlx2 polypeptide. In alternative embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (h) a NeuroD1 polypeptide and a Ctip2 polypeptide. In alternative embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (i) a NeuroD1 polypeptide, a Ascl1 polypeptide, a Dlx2 polypeptide, and a Ctip2 polypeptide. In alternative embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (j) a NeuroD1 polypeptide, a Ascl1 polypeptide, aDlx2 polypeptide, and a Isl1 polypeptide. In alternative embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (k) a NeuroD1 polypeptide, a Ascl1 polypeptide, and a Isl1 polypeptide. In alternative embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (l) a NeuroD1 polypeptide, a Ascl1 polypeptide, and a Ctip2 polypeptide. In alternative embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (m) a NeuroD1 polypeptide, a Dlx2 polypeptide, and a Ctip2 polypeptide. In alternative embodiments, the NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (n) a NeuroD1 polypeptide, a Dlx2 polypeptide, and a Ngn2 polypeptide.

In some embodiments, upon contacting the population of NG-2 cells with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the NG-2 cells stop expressing one or more glial markers selected from GFAP, Aldh1l1, S100β and Sox9. In some embodiments, the NG-2 cells stop expressing the one or more glial markers within less than 7 days, less than 6 days, less than 5 days, less than 4 days, or less than 3 days after contacting with the lipid nanoparticles.

In some embodiments, upon contacting the population of NG-2 cells with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the NG-2 cells exhibit one or more neuronal phenotypes. In some embodiments, the one or more neuronal phenotypes comprise expression of one or more neuronal markers selected from DCX, TUJ1, NeuN, and MAP2. In some embodiments, the one or more neuronal phenotypes comprise ability of firing action potentials. In some embodiments, the one or more neuronal phenotypes comprise formation of dendrites and/or exons on the cell surface. In some embodiments, the one or more neuronal phenotypes comprise formation of synapses with a neighboring cell. In some embodiments, the one or more neuronal phenotypes comprise the ability of releasing synaptic currents. In some embodiments, the synaptic currents are glutamatergic current, GABAergic current, Dopaminergic current, glycinergic current, serotonergic current or norepinephrinergic current.

In some embodiments, upon contacting the population of NG-2 cells with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the NG-2 cells are converted into functional neurons. In some embodiments, the functional neurons are selected from glutamatergic neurons, GABAergic neurons, dopaminergic neurons; motor neurons, glycinergic neurons, serotonergic neurons, norepinephrinergic neurons, and sensory neurons. In some embodiments, the NG-2 cells are converted into functional neurons less than 21 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, or less than 7 days after contacting with the lipid nanoparticles.

In some embodiments, the starting population of glial cells comprise reactive astrocytes. In some embodiments, the reactive astrocytes in the starting population express one or more glial cell markers selected from GFAP, Aldh1l1, S100β and Sox9. In some embodiments, the reactive astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding a NeuroD1 polypeptide. In alternative embodiments, the reactive astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding a NeuroD1 polypeptide and at least one second polypeptide selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof. In particular embodiments, the reactive astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (a) a NeuroD1 polypeptide alone. In alternative embodiments, the reactive astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (b) a NeuroD1 polypeptide and a Dlx2 polypeptide. In particular embodiments, the reactive astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (c) a NeuroD1 polypeptide and a Isl1 polypeptide. In particular embodiments, the reactive astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (d) a NeuroD1 polypeptide and a Ascl1 polypeptide. In particular embodiments, the reactive astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (e) a NeuroD1 polypeptide, a Dlx2 polypeptide and a Isl1 polypeptide. In particular embodiments, the reactive astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (f) a NeuroD1 polypeptide, a Ascl1 polypeptide and a Dlx2 polypeptide. In particular embodiments, the reactive astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (g) a NeuroD1 polypeptide, a Isl1 polypeptide and a Lhx3 polypeptide. In particular embodiments, the reactive astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (h) a NeuroD1 polypeptide, a Isl1 polypeptide, a Lhx3 polypeptide and a Dlx2 polypeptide. In particular embodiments, the reactive astrocytes are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (i) a NeuroD1 polypeptide, a Ascl1 polypeptide, a LMX1A polypeptide and a microRNA miR218.

In some embodiments, upon contacting the population of reactive astrocytes with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the reactive astrocytes stop expressing one or more glial markers selected from GFAP, Aldh1l1, S100β and Sox9. In some embodiments, the reactive astrocytes stop expressing the one or more glial markers within less than 7 days, less than 6 days, less than 5 days, less than 4 days, or less than 3 days after contacting with the lipid nanoparticles.

In some embodiments, upon contacting the population of reactive astrocytes with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the reactive astrocytes exhibit one or more neuronal phenotypes. In some embodiments, the one or more neuronal phenotypes comprise expression of one or more neuronal markers selected from DCX, TUJ1, NeuN, and MAP2. In some embodiments, the one or more neuronal phenotypes comprise ability of firing action potentials. In some embodiments, the one or more neuronal phenotypes comprise formation of dendrites and/or exons on the cell surface. In some embodiments, the one or more neuronal phenotypes comprise formation of synapses with a neighboring cell. In some embodiments, the one or more neuronal phenotypes comprise the ability of releasing synaptic currents. In some embodiments, the synaptic currents are glutamatergic current, GABAergic current, and Dopaminergic current.

In some embodiments, upon contacting the population of reactive astrocytes with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the reactive astrocytes are converted into functional neurons. In some embodiments, the functional neurons are selected from glutamatergic neurons, GABAergic neurons, dopaminergic neurons, and motor neurons. In some embodiments, the reactive astrocytes are converted into functional neurons less than 21 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, or less than 7 days after contacting with the lipid nanoparticles.

In some embodiments, the starting population of glial cells comprise reactive NG-2 cells. In some embodiments, the reactive NG-2 cells in the starting population express one or more glial cell markers selected from GFAP, Aldh1l1, S100β and Sox9. In some embodiments, the reactive NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding a NeuroD1 polypeptide. In alternative embodiments, the reactive NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding a NeuroD1 polypeptide and at least one second polypeptide selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof. In particular embodiments, the reactive NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (a) a NeuroD1 polypeptide alone. In alternative embodiments, the reactive NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (b) a NeuroD1 polypeptide and a Dlx2 polypeptide. In particular embodiments, the reactive NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (c) a NeuroD1 polypeptide and a Isl1 polypeptide. In particular embodiments, the reactive NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (d) a NeuroD1 polypeptide and a Ascl1 polypeptide. In particular embodiments, the reactive NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (e) a NeuroD1 polypeptide, a Dlx2 polypeptide and a Isl1 polypeptide. In particular embodiments, the reactive NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (f) a NeuroD1 polypeptide, a Ascl1 polypeptide and a Dlx2 polypeptide. In particular embodiments, the reactive NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (g) a NeuroD1 polypeptide, a Isl1 polypeptide and a Lhx3 polypeptide. In particular embodiments, the reactive NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (h) a NeuroD1 polypeptide, a Isl1 polypeptide, a Lhx3 polypeptide and a Dlx2 polypeptide. In particular embodiments, the reactive NG-2 cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (i) a NeuroD1 polypeptide, a Ascl1 polypeptide, a LMX1A polypeptide and a microRNA miR218.

In some embodiments, upon contacting the population of reactive NG-2 cells with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the reactive NG-2 cells stop expressing one or more glial markers selected from GFAP, Aldh1l1, S100β and Sox9. In some embodiments, the reactive NG-2 cells stop expressing the one or more glial markers within less than 7 days, less than 6 days, less than 5 days, less than 4 days, or less than 3 days after contacting with the lipid nanoparticles.

In some embodiments, upon contacting the population of reactive NG-2 cells with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the reactive NG-2 cells exhibit one or more neuronal phenotypes. In some embodiments, the one or more neuronal phenotypes comprise expression of one or more neuronal markers selected from DCX, TUJ1, NeuN, and MAP2. In some embodiments, the one or more neuronal phenotypes comprise ability of firing action potentials. In some embodiments, the one or more neuronal phenotypes comprise formation of dendrites and/or exons on the cell surface. In some embodiments, the one or more neuronal phenotypes comprise formation of synapses with a neighboring cell. In some embodiments, the one or more neuronal phenotypes comprise the ability of releasing synaptic currents. In some embodiments, the synaptic currents are glutamatergic current, GABAergic current, and Dopaminergic current.

In some embodiments, upon contacting the population of reactive NG-2 cells with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the reactive NG-2 cells are converted into functional neurons. In some embodiments, the functional neurons are selected from glutamatergic neurons, GABAergic neurons, dopaminergic neurons, and motor neurons. In some embodiments, the reactive NG-2 cells are converted into functional neurons less than 21 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, or less than 7 days after contacting with the lipid nanoparticles.

In some embodiments, the starting population of glial cells comprise microglial cells. In some embodiments, the starting population of glial cells comprise microglial cells. In some embodiments, the microglial cells in the starting population express the glial cell marker Sox9. In some embodiments, the microglial cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding a NeuroD1 polypeptide. In alternative embodiments, the microglial cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding a NeuroD1 polypeptide and at least one second polypeptide selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof. In particular embodiments, the microglial cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (a) a NeuroD1 polypeptide alone. In alternative embodiments, the microglial cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (b) a NeuroD1 polypeptide and a Dlx2 polypeptide. In alternative embodiments, the microglial cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (c) a NeuroD1 polypeptide and a Ascl1 polypeptide. In alternative embodiments, the microglial cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (d) a NeuroD1 polypeptide and a Math5 polypeptide. In alternative embodiments, the microglial cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (e) a NeuroD1 polypeptide and a Brn3a polypeptide. In alternative embodiments, the microglial cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (f) a NeuroD1 polypeptide and a Isl1 polypeptide. In alternative embodiments, the microglial cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (g) a NeuroD1 polypeptide, a Ascl1 polypeptide and a Dlx2 polypeptide. In alternative embodiments, the microglial cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (h) a NeuroD1 polypeptide, a Ascl1 polypeptide and a Math5 polypeptide. In alternative embodiments, the microglial cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (i) a NeuroD1 polypeptide, a Ascl1 polypeptide, and a Brn3a polypeptide. In alternative embodiments, the microglial cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (j) a NeuroD1 polypeptide, a Ascl1 polypeptide, and a Isl1 polypeptide.

In some embodiments, upon contacting the population of microglial cells with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the microglial cells stop expressing Sox9. In some embodiments, the microglial cells stop expressing the one or more glial markers within less than 7 days, less than 6 days, less than 5 days, less than 4 days, or less than 3 days after contacting with the lipid nanoparticles.

In some embodiments, upon contacting the population of microglial cells with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the microglial cells exhibit one or more neuronal phenotypes. In some embodiments, the one or more neuronal phenotypes comprise expression of one or more neuronal markers selected from Rbpms, Brn3a, NeuN, and Opsin. In some embodiments, the one or more neuronal phenotypes comprise ability of firing action potentials. In some embodiments, the one or more neuronal phenotypes comprise formation of dendrites and/or exons on the cell surface. In some embodiments, the one or more neuronal phenotypes comprise formation of synapses with a neighboring cell. In some embodiments, the one or more neuronal phenotypes comprise the ability of releasing synaptic currents.

In some embodiments, the starting population of glial cells comprise Müller cells. In some embodiments, the Müller cells in the starting population express the glial cell marker Sox9. In some embodiments, the Müller cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding a NeuroD1 polypeptide. In alternative embodiments, the Müller cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding a NeuroD1 polypeptide and at least one second polypeptide selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof. In particular embodiments, the Müller cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (a) a NeuroD1 polypeptide alone. In alternative embodiments, the Müller cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (b) a NeuroD1 polypeptide and a Dlx2 polypeptide. In alternative embodiments, the Müller cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (c) a NeuroD1 polypeptide and a Ascl1 polypeptide. In alternative embodiments, the Müller cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (d) a NeuroD1 polypeptide and a Math5 polypeptide. In alternative embodiments, the Müller cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (e) a NeuroD1 polypeptide and a Brn3a polypeptide. In alternative embodiments, the Müller cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (f) a NeuroD1 polypeptide and a Isl1 polypeptide. In alternative embodiments, the Müller cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (g) a NeuroD1 polypeptide, a Ascl1 polypeptide and a Dlx2 polypeptide. In alternative embodiments, the Müller cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (h) a NeuroD1 polypeptide, a Ascl1 polypeptide and a Math5 polypeptide. In alternative embodiments, the Müller cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (i) a NeuroD1 polypeptide, a Ascl1 polypeptide, and a Brn3a polypeptide. In alternative embodiments, the Müller cells are contacted with lipid nanoparticles comprising nucleic acid molecules encoding (j) a NeuroD1 polypeptide, a Ascl1 polypeptide, and a Isl1 polypeptide.

In some embodiments, upon contacting the population of Müller cells with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the Müller cells stop expressing Sox9. In some embodiments, the Müller cells stop expressing the one or more glial markers within less than 7 days, less than 6 days, less than 5 days, less than 4 days, or less than 3 days after contacting with the lipid nanoparticles.

In some embodiments, upon contacting the population of Müller cells with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the Müller cells exhibit one or more neuronal phenotypes. In some embodiments, the one or more neuronal phenotypes comprise expression of one or more neuronal markers selected from Rbpms, Brn3a, NeuN, and Opsin. In some embodiments, the one or more neuronal phenotypes comprise ability of firing action potentials. In some embodiments, the one or more neuronal phenotypes comprise formation of dendrites and/or exons on the cell surface. In some embodiments, the one or more neuronal phenotypes comprise formation of synapses with a neighboring cell. In some embodiments, the one or more neuronal phenotypes comprise the ability of releasing synaptic currents. In some embodiments, the one or more neuronal phenotypes comprise formation of outer segments (OS) on the cells.

In some embodiments, upon contacting the population of astrocytes with lipid nanoparticles comprising nucleic acid molecules encoding the one or more reprogramming factors comprising a NeuroD1 polypeptide, the Müller cells are converted into functional neurons. In some embodiments, the functional neurons are selected from photo-receptor cells, amacrine cells, and retinal ganglion cells. In some embodiments, the Müller cells are converted into functional neurons less than 21 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, or less than 7 days after contacting with the lipid nanoparticles.

In some embodiments, the starting population of glial cells that are converted into functional neurons by the present methods are located in vitro. In some embodiments, the starting population of glial cells that are converted into functional neurons are isolated from a subject. In some embodiments, the starting population of glial cells are in an in vitro cell culture. In some embodiments, to convert an in vitro population of glial cells into functional neurons using the present method, an effective amount a lipid nanoparticle composition comprising nucleic acid molecules encoding one or more reprograming factors comprising a NeuroD1 polypeptide according to the present disclosure is contacted with the starting population of glial cells under a suitable condition. In some embodiments, the lipid nanoparticles are added to the glial cell culture. In some embodiments, the glial cells uptake the lipid nanoparticles via endocytosis and the nucleic acid molecules are expressed by the glial cells.

In some embodiments, the starting population of glial cells that are converted into functional neurons by the present methods are located in vivo. In some embodiments, the starting population of glial cells are located in situ in a subject. In some embodiments, the starting population of glial cells are located in the peripheral nervous system of a subject. In some embodiments, the starting population of glial cells are located in the central nervous system of a subject. In some embodiments, the starting population of glial cells are located in the spinal cord of a subject.

In some embodiments, the starting population of glial cells are located in the brain of a subject. In some embodiments, the starting population of glial cells are located in the grey matter of the brain. In some embodiments, the starting population of glial cells are located in the white matter of the brain. In some embodiments, the starting population of glial cells are located in the brain striatum. In some embodiments, the starting population of glial cells are located in the cortex of the brain. In some embodiments, the starting population of glial cells are located in the hippocampus of the brain. In some embodiments, the starting population of glial cells are located in the cerebellum of the brain. In some embodiments, the starting population of glial cells comprises one or more glial cell types selected from astrocytes, reactive astrocytes, NG-2 cells, reactive NG-2 cells, and microglial cells.

In some embodiments, the starting population of glial cells are located in the eye of a subject. In some embodiments, the starting population of glial cells are surrounding the retina of the eye. In some embodiments, the starting population of glial cells are surrounding the optic nerves of the eye. In some embodiments, the starting population of glial cells comprises microglial cells. In some embodiments, the starting population of glial cells comprises Müller cells.

In some embodiments, to convert an in vitro population of glial cells into functional neurons using the present method, an effective amount a lipid nanoparticle composition comprising nucleic acid molecules encoding one or more reprograming factors comprising a NeuroD1 polypeptide according to the present disclosure is administered to the subject. In some embodiments, the administration is via local administration to the tissue or organ where the starting population of glial cells are located.

In some embodiments, upon administration, the starting population of glial cells endocytose the lipid nanoparticles and express the encoded one or more reprogramming factors comprising a NeuroD1 polypeptide.

In some embodiments, the starting population of glial cells start to undergo trans-differentiation into neurons within less than 7 days following administration of the lipid nanoparticles as described herein. In some embodiments, within less than 7 days of the administration, the starting population of glial cells reduce or stop expressing one or more glial cell markers. In some embodiments, the starting population of glial cells comprises one or more cell types selected from astrocytes, reactive astrocytes, NG-2 cells, and reactive NG-2 cells, and wherein expression of one or more glial markers selected from GFAP, Aldh1l1, S100β, and Sox9 starts to reduce from the population of cells within less than 7 days of the administration. In some embodiments, the starting population of glial cells comprises one or more cell types selected from microglial cells and Müller cells, and wherein expression Sox9 starts to reduce from the population of cells within less than 7 days of the administration.

In some embodiments, within less than 7 days of the administration, the starting population of glial cells start to express one or more immature neuron markers. In specific embodiments, the starting population of glial cells comprises one or more cell types selected from astrocytes, reactive astrocytes, NG-2 cells, and reactive NG-2 cells, and wherein expression of one or more immature neuron markers selected from DCX and TUJ1 begins within less than 7 days of the administration.

In some embodiments, within less than 14 days of the administration, matured neurons are formed from the starting population of glial cells. In specific embodiments, the starting population of glial cells comprises one or more cell types selected from astrocytes, reactive astrocytes, NG-2 cells, and reactive NG-2 cells, and matured neurons expressing one or more neuronal markers selected from NeuN and MAP2 are formed within less than 14 days of the administration. In specific embodiments, the starting population of glial cells comprises one or more cell types selected from microglial cells and Müller cells, and matured neurons expressing one or more neuronal markers selected from Rbpms, Brn3a, NeuN, and Opsin are formed within less than 14 days of the administration.

In specific embodiments, the starting population of glial cells comprises one or more cell types selected from microglial cells and Müller cells, and upon administration of the lipid nanoparticles, the cells in the population exhibit at least one structural morphology of a photoreceptor cell. In some embodiments, the cells in the population form outer segment (OS⁺) . In some embodiments, the outer segments are formed within less than 14 days of the administration.

In some embodiments, within less than 14 days of the administration, matured neurons are formed from the starting population of glial cells. In some embodiments, matured neurons exhibiting one or more neuronal morphologies are formed from the starting population of glial cells. In some embodiments, the one or more neuronal morphologies are selected from the ability of firing action potentials, formation of dendrites and/or exons on the cell surface, and formation of synapses with a neighboring cell. In some embodiments, the one or more neuronal phenotypes comprise the ability of releasing synaptic currents. In some embodiments, the synaptic currents are glutamatergic current, GABAergic current, Dopaminergic current, glycinergic current, serotonergic current or norepinephrinergic current.

In some embodiments, upon administration, the starting population of glial cells are converted in situ into functional neurons where the glial cells used to locate. In some embodiments, the converted neurons are integrated into the neuronal network in situ where the glial cells used to locate. In some embodiments, the converted neurons form synapses with neighboring neurons where the glial cells used to locate.

5.5 Nanoparticle Compositions

In one aspect, nucleic acid molecules described herein are formulated for in vitro and in vivo delivery. Particularly, in some embodiments the nucleic acid molecule is formulated into a lipid-containing composition. In some embodiments, the lipid-containing composition forms lipid nanoparticles enclosing the nucleic acid molecule within a lipid shell. In some embodiments, the lipid shells protect the nucleic acid molecules from degradation. In some embodiments, the lipid nanoparticles also facilitate transportation of the enclosed nucleic acid molecules into intracellular compartments and/or machinery to exert an intended therapeutic of prophylactic function. In certain embodiments, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease. Lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, such as those disclosed in, e.g., U.S. Patent Publication No. 2004/0142025, U.S. Patent Publication No. 2007/0042031, PCT Publication No. WO 2017/004143, PCT Publication No. WO 2015/199952, PCT Publication No. WO 2013/016058, and PCT Publication No. WO 2013/086373, the full disclosures of each of which are herein incorporated by reference in their entirety for all purposes.

In some embodiments, the largest dimension of a nanoparticle composition provided herein is 1 μm or shorter (e.g., ≤1 μm, ≤900 nm, ≤800 nm, ≤700 nm, ≤600 nm, ≤500 nm, ≤400 nm, ≤300 nm, ≤200 nm, ≤175 nm, ≤150 nm, ≤125 nm, ≤100 nm, ≤75 nm, ≤50 nm, or shorter) , such as when measured by dynamic light scattering (DLS) , transmission electron microscopy, scanning electron microscopy, or another method. In one embodiment, the lipid nanoparticle provided herein has at least one dimension that is in the range of from about 40 to about 200 nm. In one embodiment, the at least one dimension is in the range of from about 40 to about 100 nm.

Nanoparticle compositions that can be used in connection with the present disclosure include, for example, lipid nanoparticles (LNPs) , nano liproprotein particles, liposomes, lipid vesicles, and lipoplexes. In some embodiments, nanoparticle compositions are vesicles including one or more lipid bilayers. In some embodiments, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers may be functionalized and/or crosslinked to one another. Lipid bilayers may include one or more ligands, proteins, or channels.

In some embodiments, nanoparticle compositions as described comprise a lipid component including at least one lipid, such as a compound according to one of Formulae (I) to (IV) (and sub-formulas thereof) as described herein. For example, in some embodiments, a nanoparticle composition may include a lipid component including one of compounds provided herein. Nanoparticle compositions may also include one or more other lipid or non-lipid components as described below.

5.5.1 Ionizable Lipids

As described herein, in some embodiments, a nanoparticle composition provided herein comprises one or more charged or ionizable lipids in addition to a cationic lipid. Without being bound by the theory, it is contemplated that certain charged or zwitterionic lipid components of a nanoparticle composition resembles the lipid component in the cell membrane, thereby can improve cellular uptake of the nanoparticle. Exemplary charged or ionizable lipids that can form part of the present nanoparticle composition include but are not limited to ( (4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315) , 3- (didodecylamino) -N1, N1, 4-tridodecyl-1-piperazineethanamine (KL10) , N1- [2- (didodecylamino) ethyl] -N1, N4, N4-tridodecyl-1, 4-piperazinediethanamine (KL22) , 14, 25-ditridecyl-15, 18, 21, 24-tetraaza-octatriacontane (KL25) , 1, 2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA) , 2, 2-dilinoleyl-4-dimethylaminomethyl- [1, 3] -dioxolane (DLin-K-DMA) , heptatriaconta-6, 9, 28, 31-tetraen-19-yl 4- (dimethylamino) butanoate (DLin-MC3-DMA) , 2, 2-dilinoleyl-4- (2-dimethylaminoethyl) - [1, 3] -dioxolane (DLin-KC2-DMA) , 1, 2-dioleyloxy-N, N-dimethylaminopropane (DODMA) , 2- ( {8- [ (3β) -cholest-5-en-3-yloxy] octyl} oxy) -N, N-dimethyl-3 [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy] propan-1-amine (Octyl-CLinDMA) , (2R) -2- ( {8- [ (3β) -cholest-5-en-3-yloxy] octyl} oxy) -N, N-dimethyl-3- [ (9Z, 12Z) --octadeca-9, 12-dien-1-yloxy] propan-1-amine (Octyl-CLinDMA (2R) ) , (2S) -2- ( {8- [ (3β) -cholest-5-en-3-yloxy] octyl} oxy) -N, N-dimethyl-3- [ (9Z-, 12Z) -octadeca-9, 12-dien-1-yloxy] propan-1-amine (Octyl-CLinDMA (2S) ) , (12Z, 15Z) -N, N-dimethyl-2-nonylhenicosa-12, 15-den-1-amine, N, N- dimethyl-1- { (1S, 2R) -2-octylcyclopropyl} heptadecan-8-amine. Additional exemplary charged or ionizable lipids that can form part of the present nanoparticle composition include the lipids (e.g., lipid 5) described in Sabnis et al. “ANovel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates” , Molecular Therapy Vol. 26 No 6, 2018, the entirety of which is incorporated herein by reference.

In some embodiments, suitable cationic lipids include N- [1- (2, 3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) ; N- [1- (2, 3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTAP) ; 1, 2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC) ; 1, 2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC) ; 1, 2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC) ; 1, 2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14: 1) ; N1- [2- ( (1S) -1- [ (3-aminopropyl) amino] -4- [di (3-amino-propyl) amino] butylcarboxamido) ethyl] -3, 4-di [oleyloxy] -benzamide (MVL5) ; dioctadecylamido-glycylspermine (DOGS) ; 3b- [N- (N', N'-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol) ; dioctadecyldimethylammonium bromide (DDAB) ; SAINT-2, N-methyl-4- (dioleyl) methylpyridinium; 1, 2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE) ; 1, 2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE) ; 1, 2-dioleoyloxypropyl-3-dimethylhydroxyethyl ammonium chloride (DORI) ; di-alkylated amino acid (DILA²) (e.g., C18: 1-norArg-C16) ; dioleyldimethylammonium chloride (DODAC) ; 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC) ; 1, 2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC) ; (R) -5- (dimethylamino) pentane-1, 2-diyl dioleate hydrochloride (DODAPen-Cl) ; (R) -5-guanidinopentane-1, 2-diyl dioleate hydrochloride (DOPen-G) ; and (R) -N, N, N-trimethyl-4, 5-bis (oleoyloxy) pentan-1-aminium chloride (DOTAPen) . Also suitable are cationic lipids with headgroups that are charged at physiological pH, such as primary amines (e.g., DODAG N', N'-dioctadecyl-N-4, 8-diaza-10-aminodecanoylglycine amide) and guanidinium head groups (e.g., bis-guanidinium-spermidine-cholesterol (BGSC) , bis-guanidiniumtren-cholesterol (BGTC) , PONA, and (R) -5-guanidinopentane-1, 2-diyl dioleate hydrochloride (DOPen-G) ) . Yet another suitable cationic lipid is (R) -5- (dimethylamino) pentane-1, 2-diyl dioleate hydrochloride (DODAPen-Cl) . In certain embodiments, the cationic lipid is a particular enantiomer or the racemic form, and includes the various salt forms of a cationic lipid as above (e.g., chloride or sulfate) . For example, in some embodiments, the cationic lipid is N- [1- (2, 3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTAP-Cl) or N- [1- (2, 3-dioleoyloxy) propyl] -N, N, N-trimethylammonium sulfate (DOTAP-Sulfate) . In some embodiments, the cationic lipid is an ionizable cationic lipid such as, e.g., dioctadecyldimethylammonium bromide (DDAB) ; 1, 2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA) ; 2, 2-dilinoleyl-4- (2dimethylaminoethyl) - [1, 3] -dioxolane (DLin-KC2-DMA) ; heptatriaconta-6, 9, 28, 31-tetraen-19-yl 4- (dimethylamino) butanoate (DLin-MC3-DMA) ; 1, 2-dioleoyloxy-3-dimethylaminopropane (DODAP) ; 1, 2-dioleyloxy-3- dimethylaminopropane (DODMA) ; and morpholinocholesterol (Mo-CHOL) . In certain embodiments, a lipid nanoparticle includes a combination or two or more cationic lipids (e.g., two or more cationic lipids as above) .

Additionally, in some embodiments, the charged or ionizable lipid that can form part of the present nanoparticle composition is a lipid including a cyclic amine group. Additional cationic lipids that are suitable for the formulations and methods disclosed herein include those described in WO2015199952, WO2016176330, and WO2015011633, the entire contents of each of which are hereby incorporated by reference in their entireties. Additionally, in some embodiments, the charged or ionizable lipid that can form part of the present nanoparticle composition is a lipid including a cyclic amine group. Additional cationic lipids that are suitable for the formulations and methods disclosed herein include those described in WO2015199952, WO2016176330, WO2015011633, WO2018/081480, the entire contents of each of which are hereby incorporated by reference in their entireties.

5.5.2 Polymer Conjugated Lipids

In some embodiments, the lipid component of a nanoparticle composition can include one or more polymer conjugated lipids, such as PEGylated lipids (PEG lipids) . Without being bound by the theory, it is contemplated that a polymer conjugated lipid component in a nanoparticle composition can improve of colloidal stability and/or reduce protein absorption of the nanoparticles. Exemplary cationic lipids that can be used in connection with the present disclosure include but are not limited to 2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide (ALC-0159) , PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modifieddialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, Ceramide-PEG2000, or Chol-PEG2000.

In one embodiment, the polymer conjugated lipid is a pegylated lipid. For example, some embodiments include a pegylated diacylglycerol (PEG-DAG) such as 1- (monomethoxy-polyethyleneglycol) -2, 3-dimyristoylglycerol (PEG-DMG) , a pegylated phosphatidylethanoloamine (PEG-PE) , a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O- (2’, 3’-di (tetradecanoyloxy) propyl-1-O- (ω-methoxy (polyethoxy) ethyl) butanedioate (PEG-S-DMG) , a pegylated ceramide (PEG-cer) , or a PEG dialkoxypropylcarbamate such as ω-methoxy (polyethoxy) ethyl-N- (2, 3-di (tetradecanoxy) propyl) carbamate or 2, 3-di (tetradecanoxy) propyl-N- (ω-methoxy (polyethoxy) ethyl) carbamate.

In one embodiment, the polymer conjugated lipid is present in a concentration ranging from 1.0 to 2.5 molar percent. In one embodiment, the polymer conjugated lipid is present in a concentration of about 1.7 molar percent. In one embodiment, the polymer conjugated lipid is present in a concentration of about 1.5 molar percent.

In one embodiment, the molar ratio of cationic lipid to the polymer conjugated lipid ranges from about 35: 1 to about 25: 1. In one embodiment, the molar ratio of cationic lipid to polymer conjugated lipid ranges from about 100: 1 to about 20: 1.

In one embodiment, the pegylated lipid has the following Formula:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

R¹² and R¹³ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.

In one embodiment, R¹² and R¹³ are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In other embodiments, the average w ranges from 42 to 55, for example, the average w is 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55. In some specific embodiments, the average w is about 49.

In one embodiment, the pegylated lipid has the following Formula:

wherein the average w is about 49.

5.5.3 Structural Lipids

In some embodiments, the lipid component of a nanoparticle composition can include one or more structural lipids. Without being bound by the theory, it is contemplated that structural lipids can stabilize the amphiphilic structure of a nanoparticle, such as but not limited to the lipid bilayer structure of a nanoparticle. Exemplary structural lipids that can be used in connection with the present disclosure include but are not limited to cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) , or a combination thereof.

In one embodiment, the lipid nanoparticles provided herein comprise a steroid or steroid analogue. In one embodiment, the steroid or steroid analogue is cholesterol. In one embodiment, the steroid is present in a concentration ranging from 39 to 49 molar percent, 40 to 46 molar percent, from 40 to 44 molar percent, from 40 to 42 molar percent, from 42 to 44 molar percent, or from 44 to 46 molar percent. In one embodiment, the steroid is present in a concentration of 40, 41, 42, 43, 44, 45, or 46 molar percent.

In one embodiment, the molar ratio of cationic lipid to the steroid ranges from 1.0: 0.9 to 1.0: 1.2, or from 1.0: 1.0 to 1.0: 1.2. In one embodiment, the molar ratio of cationic lipid to cholesterol ranges from about 5: 1 to 1: 1. In one embodiment, the steroid is present in a concentration ranging from 32 to 40 mol percent of the steroid.

5.5.4 Phospholipids

In some embodiments, the lipid component of a nanoparticle composition can include one or more phospholipids, such as one or more (poly) unsaturated lipids. Without being bound by the theory, it is contemplated that phospholipids may assemble into one or more lipid bilayers structures. Exemplary phospholipids that can form part of the present nanoparticle composition include but are not limited to 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) , 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) , 1, 2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) , 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC) , 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) , 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) , 1, 2-diundecanoyl-sn-glycero-phosphocholine (DUPC) , 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) , 1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18: 0 Diether PC) , 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC) , 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) , 1, 2-dilinolenoyl-sn-glycero-3-phosphocholine, 1, 2-diarachidonoyl-sn-glycero-3-phosphocholine, 1, 2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1, 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE) , 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1, 2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1, 2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG) , and sphingomyelin. In certain embodiments, a nanoparticle composition includes DSPC. In certain embodiments, a nanoparticle composition includes DOPE. In some embodiments, a nanoparticle composition includes both DSPC and DOPE.

Additional exemplary neutral lipids include, for example, dipalmitoylphosphatidylglycerol (DPPG) , palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1carboxylate (DOPE-mal) , dipalmitoyl phosphatidyl ethanolamine (DPPE) , dimyristoylphosphoethanolamine (DMPE) , distearoyl-phosphatidylethanolamine (DSPE) , 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanol amine (SOPE) , and 1, 2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE) . In one embodiment, the neutral lipid is 1, 2-distearoyl-sn-glycero-3phosphocholine (DSPC) . In one embodiment, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

In one embodiment, the neutral lipid is phosphatidylcholine (PC) , phosphatidylethanolamine (PE) phosphatidylserine (PS) , phosphatidic acid (PA) , or phosphatidylglycerol (PG) .

Additionally phospholipids that can form part of the present nanoparticle composition also include those described in WO2017/112865, the entire content of which is hereby incorporated by reference in its entirety.

5.5.5 Formulation

According to the present disclosure, nanoparticle compositions described herein can include at least one lipid component and one or more additional components, such as a therapeutic and/or prophylactic agent (e.g., the therapeutic nucleic acid described herein) . A nanoparticle composition may be designed for one or more specific applications or targets. The elements of a nanoparticle composition may be selected based on a particular application or target, and/or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements. Similarly, the particular formulation of a nanoparticle composition may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combinations of elements.

The lipid component of a nanoparticle composition may include, for example, a cationic lipid (e.g., ALC-0315) , a phospholipid (such as an unsaturated lipid, e.g., DOPE or DSPC) , a PEG lipid, and a structural lipid. The elements of the lipid component may be provided in specific fractions.

In one embodiment, provided herein is a nanoparticle composition comprising a cationic or ionizable lipid compound provided herein, a nucleic acid molecule, and one or more excipients. In one embodiment, cationic or ionizable lipid compound comprises one or more ionizable lipid compounds described herein. In one embodiment, the one or more excipients are selected from phospholipids, steroids, and polymer conjugated lipids. In one embodiment, the therapeutic agent is encapsulated within or associated with the lipid nanoparticle.

In one embodiment, provided herein is a nanoparticle composition (lipid nanoparticle) comprising:

i) between about 20 and about 60 mol percent of an ionizable lipid;

ii) between about 5 and about 30 mol percent of a phospholipid;

iii) between about 25 and about 70 mol percent of a steroid;

(vi) between about 0.2 and about 10 mol percent of a PEG-conjugated lipid.

i) between about 30 and about 50 mol percent of an ionizable lipid;

ii) between about 5 and about 20 mol percent of a phospholipid;

iii) between about 40 and about 60 mol percent of a steroid;

iv) between about 1 and about 5 mol percent of a PEG-conjugated lipid

i) between about 40 and about 50 mol percent of an ionizable lipid;

ii) between about 5 and about 10 mol percent of a phospholipid;

iii) between about 40 and about 50 mol percent of a steroid;

iv) between about 1 and about 2 mol percent of a PEG-conjugated lipid.

In certain embodiments, provided herein is a nanoparticle composition (lipid nanoparticle) comprising:

i) about 46.30 mol percent of an ionizable lipid;

ii) about 9.40 mol percent of an phospholipid;

iii) about 42.70 mol percent of a steroid; and

iv) about 1.60 mol percent of a PEG-conjugated lipid.

In specific embodiments, the ionizable lipid is ( (4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315) . In specific embodiments, the phospholipid is DSPC. In specific embodiments, the steroid is cholesterol. In specific embodiments, the PEG conjugated lipid is 2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide (ALC-0159) .

i) between 40 and 50 mol percent of an ionizable lipid;

ii) a neutral lipid;

iii) a steroid;

iv) a polymer conjugated lipid; and

v) a nucleic acid molecule.

As used herein, “mol percent” refers to a component’s molar percentage relative to total mols of all lipid components in the LNP (i.e., total mols of cationic lipid (s) , the neutral lipid, the steroid and the polymer conjugated lipid) .

In one embodiment, the lipid nanoparticle comprises from 41 to 49 mol percent, from 41 to 48 mol percent, from 42 to 48 mol percent, from 43 to 48 mol percent, from 44 to 48 mol percent, from 45 to 48 mol percent, from 46 to 48 mol percent, or from 47.2 to 47.8 mol percent of the cationic lipid. In one embodiment, the lipid nanoparticle comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9 or 48.0 mol percent of the ionizable lipid.

In one embodiment, the neutral lipid is present in a concentration ranging from 5 to 15 mol percent, 7 to 13 mol percent, or 9 to 11 mol percent. In one embodiment, the neutral lipid is present in a concentration of about 9.5, 10 or 10.5 mol percent. In one embodiment, the molar ratio of the cationic lipid to the neutral lipid ranges from about 4.1: 1.0 to about 4.9: 1.0, from about 4.5: 1.0 to about 4.8: 1.0, or from about 4.7: 1.0 to 4.8: 1.0.

In one embodiment, the steroid is present in a concentration ranging from 39 to 49 molar percent, 40 to 46 molar percent, from 40 to 44 molar percent, from 40 to 42 molar percent, from 42 to 44 molar percent, or from 44 to 46 molar percent. In one embodiment, the steroid is present in a concentration of 40, 41, 42, 43, 44, 45, or 46 molar percent. In one embodiment, the molar ratio of cationic lipid to the steroid ranges from 1.0: 0.9 to 1.0: 1.2, or from 1.0: 1.0 to 1.0: 1.2. In one embodiment, the steroid is cholesterol.

In one embodiment, the therapeutic agent to lipid ratio in the LNP (i.e., N/P, were N represents the moles of cationic lipid and P represents the moles of phosphate present as part of the nucleic acid backbone) range from 2: 1 to 30: 1, for example 3: 1 to 22: 1. In one embodiment, N/P ranges from 6: 1 to 20: 1 or 2: 1 to 12: 1. Exemplary N/P ranges include about 3: 1. About 6: 1, about 12: 1 and about 22:1.

In one embodiment, provided herein is a lipid nanoparticle comprising:

i) a cationic lipid having an effective pKa greater than 6.0; ii) from 5 to 15 mol percent of a neutral lipid;

iii) from 1 to 15 mol percent of an anionic lipid;

iv) from 30 to 45 mol percent of a steroid;

v) a polymer conjugated lipid; and

vi) a nucleic acid molecule,

wherein the mol percent is determined based on total mol of lipid present in the lipid nanoparticle.

In one embodiment, the cationic lipid can be any of a number of lipid species which carry a net positive charge at a selected pH, such as physiological pH. Exemplary cationic lipids are described herein below. In one embodiment, the cationic lipid has a pKa greater than 6.25. In one embodiment, the cationic lipid has a pKa greater than 6.5. In one embodiment, the cationic lipid has a pKa greater than 6.1, greater than 6.2, greater than 6.3, greater than 6.35, greater than 6.4, greater than 6.45, greater than 6.55, greater than 6.6, greater than 6.65, or greater than 6.7.

In one embodiment, the lipid nanoparticle comprises from 40 to 45 mol percent of the cationic lipid. In one embodiment, the lipid nanoparticle comprises from 45 to 50 mole percent of the cationic lipid.

In one embodiment, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2: 1 to about 8: 1. In one embodiment, the lipid nanoparticle comprises from 5 to 10 mol percent of the neutral lipid.

Exemplary anionic lipids include, but are not limited to, phosphatidylglycerol, dioleoylphosphatidylglycerol (DOPG) , dipalmitoylphosphatidylglycerol (DPPG) or 1, 2-distearoyl-sn-glycero-3-phospho- (1'-rac-glycerol) (DSPG) .

In one embodiment, the lipid nanoparticle comprises from 1 to 10 mole percent of the anionic lipid. In one embodiment, the lipid nanoparticle comprises from 1 to 5 mole percent of the anionic lipid. In one embodiment, the lipid nanoparticle comprises from 1 to 9 mole percent, from 1 to 8 mole percent, from 1 to 7 mole percent, or from 1 to 6 mole percent of the anionic lipid. In one embodiment, the mol ratio of anionic lipid to neutral lipid ranges from 1: 1 to 1: 10.

In one embodiment, the steroid cholesterol. In one embodiment, the molar ratio of the cationic lipid to cholesterol ranges from about 5: 1 to 1: 1. In one embodiment, the lipid nanoparticle comprises from 32 to 40 mol percent of the steroid.

In one embodiment, the sum of the mol percent of neutral lipid and mol percent of anionic lipid ranges from 5 to 15 mol percent. In one embodiment, wherein the sum of the mol percent of neutral lipid and mol percent of anionic lipid ranges from 7 to 12 mol percent.

In one embodiment, the mol ratio of anionic lipid to neutral lipid ranges from 1: 1 to 1: 10. In one embodiment, the sum of the mol percent of neutral lipid and mol percent steroid ranges from 35 to 45 mol percent.

In one embodiment, the lipid nanoparticle comprises:

i) from 45 to 55 mol percent of the cationic lipid;

ii) from 5 to 10 mol percent of the neutral lipid;

iii) from 1 to 5 mol percent of the anionic lipid; and

iv) from 32 to 40 mol percent of the steroid.

In one embodiment, the lipid nanoparticle comprises from 1.0 to 2.5 mol percent of the conjugated lipid. In one embodiment, the polymer conjugated lipid is present in a concentration of about 1.5 mol percent.

In one embodiment, the steroid is cholesterol. In some embodiments, the steroid is present in a concentration ranging from 39 to 49 molar percent, 40 to 46 molar percent, from 40 to 44 molar percent, from 40 to 42 molar percent, from 42 to 44 molar percent, or from 44 to 46 molar percent. In one embodiment, the steroid is present in a concentration of 40, 41, 42, 43, 44, 45, or 46 molar percent. In certain embodiments, the molar ratio of cationic lipid to the steroid ranges from 1.0: 0.9 to 1.0: 1.2, or from 1.0: 1.0 to 1.0: 1.2.

In one embodiment, the molar ratio of cationic lipid to steroid ranges from 5: 1 to 1: 1.

In one embodiment, the molar ratio of cationic lipid to polymer conjugated lipid ranges from about 100: 1 to about 20: 1. In one embodiment, the molar ratio of cationic lipid to the polymer conjugated lipid ranges from about 35: 1 to about 25: 1.

In one embodiment, the lipid nanoparticle has a mean diameter ranging from 50 nm to 100 nm, or from 60 nm to 85 nm.

In one embodiment, the composition comprises a cationic lipid provided herein, DSPC, cholesterol, and PEG-lipid, and mRNA. In one embodiment, the cationic lipid provided herein, DSPC, cholesterol, and PEG-lipid are at a molar ratio of about 50: 10: 38.5: 1.5.

Nanoparticle compositions can be designed for one or more specific applications or targets. For example, a nanoparticle composition can be designed to deliver a therapeutic and/or prophylactic agent such as an RNA to a particular cell, tissue, organ, or system or group thereof in a mammal’s body. Physiochemical properties of nanoparticle compositions can be altered in order to increase selectivity for particular bodily targets. For instance, particle sizes can be adjusted based on the fenestration sizes of different organs. The therapeutic and/or prophylactic agent included in a nanoparticle composition can also be selected based on the desired delivery target or targets. For example, a therapeutic and/or prophylactic agent can be selected for a particular indication, condition, disease, or disorder and/or for delivery to a particular cell, tissue, organ, or system or group thereof (e.g., localized or specific delivery) . In certain embodiments, a nanoparticle composition can include an mRNA encoding a polypeptide of interest capable of being translated within a cell to produce the polypeptide of interest. Such a composition can be designed to be specifically delivered to a particular organ. In certain embodiments, a composition can be designed to be specifically delivered to a mammalian liver.

The amount of a therapeutic and/or prophylactic agent in a nanoparticle composition can depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the therapeutic and/or prophylactic agent. For example, the amount of an RNA useful in a nanoparticle composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of a therapeutic and/or prophylactic agent and other elements (e.g., lipids) in a nanoparticle composition can also vary. In some embodiments, the wt/wt ratio of the lipid component to a therapeutic and/or prophylactic agent in a nanoparticle composition can be from about 5: 1 to about 60: 1, such as about 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11: 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 22: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, and 60: 1. For example, the wt/wt ratio of the lipid component to a therapeutic and/or prophylactic agent can be from about 10: 1 to about 40: 1. In certain embodiments, the wt/wt ratio is about 20: 1. The amount of a therapeutic and/or prophylactic agent in a nanoparticle composition can, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy) .

In some embodiments, a nanoparticle composition includes one or more RNAs, and the one or more RNAs, lipids, and amounts thereof can be selected to provide a specific N: P ratio. The N: P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an RNA. In some embodiments, a lower N: P ratio is selected. The one or more RNA, lipids, and amounts thereof can be selected to provide an N: P ratio from about 2: 1 to about 30: 1, such as 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 12: 1, 14: 1, 16: 1, 18: 1, 20: 1, 22: 1, 24: 1, 26: 1, 28: 1, or 30: 1. In certain embodiments, the N: P ratio can be from about 2: 1 to about 8: 1. In other embodiments, the N: P ratio is from about 5: 1 to about 8: 1. For example, the N: P ratio may be about 5.0: 1, about 5.5: 1, about 5.67: 1, about 6.0: 1, about 6.5: 1, or about 7.0: 1. For example, the N: P ratio may be about 5.67: 1.

The physical properties of a nanoparticle composition can depend on the components thereof. For example, a nanoparticle composition including cholesterol as a structural lipid can have different characteristics compared to a nanoparticle composition that includes a different structural lipid. Similarly, the characteristics of a nanoparticle composition can depend on the absolute or relative amounts of its components. For instance, a nanoparticle composition including a higher molar fraction of a phospholipid may have different characteristics than a nanoparticle composition including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition.

Nanoparticle compositions may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem, Worcestershire, UK) may also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

In various embodiments, the mean size of a nanoparticle composition can be between 10s of nm and 100s of nm. For example, the mean size can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the mean size of a nanoparticle composition can be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In certain embodiments, the mean size of a nanoparticle composition can be from about 70 nm to about 100 nm. In some embodiments, the mean size can be about 80 nm. In other embodiments, the mean size can be about 100 nm.

A nanoparticle composition can be relatively homogenous. A polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition can have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a nanoparticle composition can be from about 0.10 to about 0.20.

The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species can interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a nanoparticle composition can be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

The efficiency of encapsulation of a therapeutic and/or prophylactic agent describes the amount of therapeutic and/or prophylactic agent that is encapsulated or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%) . The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and/or prophylactic agent in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and/or prophylactic agent (e.g., RNA) in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic and/or prophylactic agent can be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

A nanoparticle composition can optionally comprise one or more coatings. For example, a nanoparticle composition can be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including a composition described herein can have any useful size, tensile strength, hardness, or density.

6. EXAMPLES

The examples in this section (i.e., Section 6) are offered by way of illustration, and not by way of limitation.

6.1 Example 1: RNA design and Production.

6.1.1 mRNA design and production.

The mRNA molecules were generated via in vitro transcription of a DNA template plasmid, followed by further in vitro modifications and purification. Particularly, template plasmids contained a plasmid backbone for plasmid amplification within a bacterial host, elements required for in vitro transcription, and coding sequences (e.g., encoding a neural transcription factors or a signal polypeptide such as GFP) . To increase the protein expression level, the plasmids also comprise of UTRs (untranslated region) at the 5’ and/or 3’ ends of the coding sequence. After or during in vitro transcription, the 5’ end of transcripts were modified with a cap structure and the poly A sequence was added to the 3’ end. The mRNAs used for in vitro transfection or in vivo injection contained a 5’-UTR, a coding sequence, a 3’-UTR, a poly A sequence and with 5’ cap.

6.1.2 Circular RNA (cirRNA) design and production

Circular RNA molecules were generated via in vitro transcription of a DNA template plasmid, followed by in vitro circularization of the linear precursor RNA molecule and purification of the circular RNA. Template plasmids comprise of a plasmid backbone, which is needed for plasmid amplification within bacteria, coding sequences of neural transcription factors or GFP, elements required for in vitro transcription (T7 promoter) , for translation (IRES) , for forming circular RNA (group I intron PIE) , and for improving circularization efficiency (homologous arms and spacers) . cirRNAs for in vitro transfection or in vivo injection comprised of coding sequences of neural transcription factor or GFP or transcription factor and GFP, and upstream translational initiation sequences (IRES) , and other UTRs.

6.2 Example 2: Trans-differentiation of cultured cells induced by expression of NeuroD1.

Murine or human primary astrocytes were cultured using an in vitro cell culture system following standard protocols. Purified mRNA or cirRNA encoding the neural transcription factor (NeuroD1) was transfected into the cultured cells. Cellular expression of the NeuroD1 from the mRNA or cirRNA was detected using a fluorescently labeled antibody that specifically bound to NeuroD1. Neurons converted from astrocytes were identified by detecting the expression of neuron specific proteins, including early neuronal marker such as TUBB3, DCX, and mature neuronal marker proteins such as NeuN and MAP2.

FIG. 1 shows the expression of the neural transcription factor NeuroD1 (red) in rat primary astrocytes 24 hours post transfection of the NeuroD1-encoding mRNA. Astrocytes were labeled with green staining of glia fibrillary acidic protein (GFAP) and all cell nuclei were labeled with DAPI (blue) .

FIGS. 4A and 4B show astrocytes were converted into neurons after transfection with mRNA encoding NeuroD1. Astrocytes were labeled with green (GFAP+) ; all cell nuclei were labeled with DAPI (blue) ; neuron marker TUJ1 was labeled in red. FIG. 4A shows 6 days post transfection with NeuroD1-encoding cirRNA, rat primary astrocytes underwent trans-differentiation into neurons, as demonstrated by the expression of immature neuron marker TUJ1 (red) in the cells. As shown in FIG. 4B, 10 days post transfection with NeuroD1-encoding cirRNA, astrocytes were converted into neurons, as demonstrated by NeuN+ (red) and MAP2+ (green) positive staining. DAPI (blue) stain showed all cell nuclei.

These data indicate that the cultured murine primary astrocytes underwent morphological changes after transfection of NeuroD1-encoding mRNA or cirRNA, and demonstrated neuronal morphology by expressing early immature neuronal marker proteins such as DCX (doublecortin) , TUJ1 (class III beta tubulin) positive. At 8-12 days post transfection, converted cells were positive for mature neuronal marker protein NeuN (neuronal specific nuclear protein) , Map2 (microtubule associated protein 2) , indicating the astrocytes continued to differentiate into matured neurons.

6.3 Example 3: In vivo trans-differentiation of astrocytes induced by expression of NeuroD1

Synthesized and purified cirRNA or mRNA molecules encoding neural transcription factor NeuroD1 (NeuroD1-RNA) were encapsulated using microfluidic mixing technology. Suitable nitrogen-to-phosphorus ratios were screened and selected for different RNA sizes (molar ratio of cationic lipids: nucleic acid in the range of about 3: 1 to 8: 1) . Lipids composition containing an ionizable lipid, helper phospholipid, cholesterol, and a PEG-modified lipid were dissolved in ethanol in the molar ratios of 43: 9: 40: 1.5 in ethanol. RNA was dissolved in an acidic buffer (pH 4.0) . The two phases containing lipids and RNA molecules were mixed rapidly, followed by diluting the ethanol phase, the solubility of the lipids decreased, and the solidification was gradually precipitated in the mixed solution and lipid nanoparticles were formed with RNAs efficiently encapsulated. The residual ethanol was then removed by buffer membrane ultrafiltration. Buffer pH was neutralized. Finally, LNP/NeuroD1-RNA was concentrated, and its particle size, potential, encapsulation rate, RNA integrity and other indicators were assessed.

A LNP composition containing mRNA molecules encoding NeuroD1 (LNP/NeuroD1-mRNA) or both NeuroD1 and GFP connected by a P2A peptide (LNP/NeuroD1-p2A-GFP-mRNA) was delivered into brain striatum of mice by stereotactic injection. The mouse brains were harvested, fixed, sectioned for immunohistochemistry analysis (fluorescence) at 1 day, 4 days, 8 days, 14 days post injection to examine NeuroD1 expression and conversion of brain cells.

FIGS. 5A and 5B show in vivo neural transcription factor NeuroD1 protein expression 24 hours after injection of LNP composition containing NeuroD1 encoding RNA. NeuroD1 protein expression was shown in purple, GFP protein was shown in green, astrocyte marker (GFAP+) was shown in red and all nuclei (DAPI+) were shown in blue. Particularly, brain striatum (AP+1.0, ML+1.5, DV-3.5) of 6-8 weeks old C57 BL/6J mice were injected with 1μL solution containing LNP/NeuroD1-mRNA and a AAV9 vector encoding GFP on left side of brain and injected 1μL solution containing LNP/NeuroD1-p2A-GFP-mRNA on the right side of brain. In FIG. 5A, NeuroD1 protein expression was shown as purple labeling. AAV9-GFP was used as a tracking control on left side of the brain. As shown, NeuroD1 expression was clearly detected 24 hours after injection of LNP/ND1-mRNA or LNP/ND1-p2A-GFP-mRNA. FIG. 5B shows NeuroD1 staining (in purple) and astrocytes in red (GFAP+) under a higher magnification. All nuclei (DAPI+) show in blue. NeuroD1 expression in astrocytes were indicated by white arrows.

FIG. 6 shows in vivo conversion of astrocytes to neurons 14 days after injection of LNP/NeuroD1-p2A-GFP-mRNA. Green marks ND1-p2A-GFP-mRNA transfected cells, astrocytes are labeled red (GFAP) , mature neurons are labeled purple (NeuN) . All nuclei (DAPI+) show in blue. Particularly, striatum (AP+1.0, ML+1.5, DV-3.5) of 6-8 weeks old C57 BL/6J mice were injected 1μL solution of LNP/NeuroD1-p2A-GFP-mRNA on one side and injected with 1μL LNP/GFP-mRNA on the other side. As shown in FIG. 6A, 14 days post injection, astrocytes underwent trans-differentiation into neurons, as demonstrated by the expression of NeuroD1 (GFP+) and the neuronal marker NeuN protein (NeuN+) in the cells that also exhibited a typical neuron morphology. FIG. 6B shows the other side striatum of the same rat brain as in FIG. 6A, in which LNP/GFP-mRNA was injected. As shown, 14 days post LNP/GFP-mRNA injection, no green labeled cell showing neuron morphology was observed which indicates there was no astrocyte to neuron conversion here.

These data indicate that in vivo delivered NeuroD1 encoding mRNA enabled NeuroD1 expression in astrocytes shown by NeuroD1 positive staining 24 hours post injection and induced astrocyte-to-neuron conversion as shown by neural marker NeuN positive staining in LNP/ND1-p2A-GFP-mRNA transfected cells (GFP+) 14 days post injection.

6.4 Example 4: Trans-differentiation of cultured cells induced by expression of two transcription protein factors (NeuroD1 and Ascl1) .

Murine primary astrocytes were cultured using an in vitro cell culture system following standard protocols. Purified mRNA encoding the neural transcription factor (NeuroD1 and Ascl1) was transfected into the cultured cells. Cellular expression of the NeuroD1 and Ascl1 from the mRNA was detected using a fluorescently labeled antibody that specifically bound to the two proteins, respectively. Neurons converted from astrocytes were identified by detecting the expression of neuron specific proteins, including mature neuronal marker proteins such as NeuN and MAP2.

FIG. 10 shows neurons generated by trans-differentiation of rat primary astrocytes. The generated neurons showed NeuN-positive (left panel, red) and MAP2-positive (middle panel, green) staining 3 weeks after co-transfection of the rat primary astrocytes with the NeuroD1-encoding mRNA and the Ascl1-encoding mRNA. DAPI stained all cell nuclei in blue (right panel, all color) .

6.5 Example 5: Trans-differentiation of cultured cells induced by expression of two transcription protein factors (NeuroD1 and Dlx2) .

Murine primary astrocytes were cultured using an in vitro cell culture system following standard protocols. Purified mRNA encoding the neural transcription factor (NeuroD1 and Dlx2) was transfected into the cultured cells. Cellular expression of the NeuroD1 and Ascl1 from the mRNA was detected using a fluorescently labeled antibody that specifically bound to the two proteins, respectively. Neurons converted from astrocytes were identified by detecting the expression of neuron specific proteins, including mature neuronal marker proteins such as NeuN and MAP2.

6.6 Example 6: Trans-differentiation of cultured cells induced by expression of two transcription protein factors (Dlx2 and Ascl1) .

Murine primary astrocytes were cultured using an in vitro cell culture system following standard protocols. Purified mRNA encoding the neural transcription factor (Dlx2 and Ascl1) was transfected into the cultured cells. Cellular expression of the Dlx2 and Ascl1 from the mRNA was detected using a fluorescently labeled antibody that specifically bound to the two proteins, respectively. Neurons converted from astrocytes were identified by detecting the expression of neuron specific proteins, including mature neuronal marker proteins such as NeuN and MAP2.

FIG. 17 shows neurons generated by trans-differentiation of rat primary astrocytes. The generated neurons showed NeuN-positive (left panel, red) and MAP2-positive (middle panel, green) staining 2 weeks after co-transfection of the rat primary astrocytes with the Ascl1-encoding mRNA and the Dlx2-encoding mRNA.DAPI stained all cell nuclei in blue (right panel, all color) .

Claims

A lipid nanoparticle composition comprising a nucleic acid molecule and at least one lipid, wherein the nucleic acid molecule comprises an expression sequence encoding a NeuroD1 polypeptide; and wherein the at least one lipid forms lipid nanoparticles encompassing the nucleic acid molecule.
The lipid nanoparticle composition of claim 1, wherein the expression sequence is a multi-cistronic sequence encoding the NeuroD1 polypeptide and at least one second polypeptide selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof.
The lipid nanoparticle composition of claim 2, wherein the at least one second polypeptide comprises two polypeptides each independently selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, and Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof.
The lipid nanoparticle composition of claim 2, wherein the expression sequence encodes:

(a) the NeuroD1 polypeptide;

(b) the NeuroD1 polypeptide and the Dlx2 polypeptide;

(c) the NeuroD1 polypeptide and the Isl1 polypeptide;

(d) the NeuroD1 polypeptide and the Ascl1 polypeptide;

(e) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Isl1 polypeptide;

(f) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ascl1 polypeptide;

(g) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ngn2 polypeptide;

(h) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ctip2 polypeptide;

(i) the NeuroD1 polypeptide, the Isl1 polypeptide, and the Ascl1 polypeptide;

(j) the NeuroD1 polypeptide, the Ils1 polypeptide, and the Lhx3 polypeptide;

(k) the NeuroD1 polypeptide, the Ascl1 polypeptide, and the Ctip2 polypeptide;

(l) the NeuroD1 polypeptide, the Dlx2 polypeptide, the Ascl1 polypeptide, and the Isl1 polypeptide; or

(m) the NeuroD1 polypeptide, the Dlx2 polypeptide, the Ascl1 polypeptide, and the Ctip2 polypeptide.
The lipid nanoparticle composition of any one of claims 2 to 4, wherein the expression sequence comprises at least one ribosomal skipping element located between sequences encoding the NeuroD1 polypeptide and the at least one second polypeptide.
The lipid nanoparticle composition of claim 5, wherein the ribosomal skipping element encodes a proteasome cleavage site selected from thosea-asigna virus 2A peptide (T2A) , porcine teschovirus-1 2 A peptide (P2A) , foot-and-mouth disease virus 2 A peptide (F2A) , equine rhinitis A vims 2A peptide (E2A) , cytoplasmic polyhedrosis vims 2A peptide (BmCPV 2A) , or flacherie vims of B. mori 2A peptide (BmIFV 2A) .
The lipid nanoparticle composition of any one of claims 1 to 6, wherein the nucleic acid molecule is a linear mRNA molecule, and wherein the expression sequence comprises one or more open reading frames (ORFs) , and wherein at least one of the ORFs encodes the NeuroD1 polypeptide.
The lipid nanoparticle composition of claim 7, wherein the expression sequence further comprises a 5’ untranslated region (5’-UTR) upstream to the open reading frame and/or a 3’-UTR downstream of the open reading frame.
The lipid nanoparticle composition of claim 8, wherein the 5’-UTR comprises an IRES.
The lipid nanoparticle composition of claim 8, wherein the 5’-UTR comprises the sequence selected from the group consisting of SEQ ID NOS: 53 and 55; optionally wherein the 3’-UTR comprises the sequence selected from the group consisting of SEQ ID NOS: 57 and 59; optionally wherein the 5’-UTR comprises a 5’-cap structure; optionally wherein the 3’-UTR further comprises a polyA region of about 60 to about 200 residues in length.
The lipid nanoparticle composition of any one of claims 1 to 10, wherein the encoded NeuroD1 polypeptide is human NeuroD1 or a functional derivative thereof; wherein optionally, the functional variant of human NeuroD1 comprises an amino acid sequence that has at least about 90%, at least about 95%, at least about 97%, or at least about 99%sequence identity to SEQ ID NO:1; wherein optionally, the functional variant of human NeuroD1 comprises an amino acid sequence that has at least about 90%, at least about 95%, at least about 97%, or at least about 99%sequence identity to SEQ ID NO: 2.
The lipid nanoparticle composition of any one of claims 1 to 10, wherein the expression sequence comprises a coding sequence for the NeuroD1 polypeptide having:

(a) the DNA sequence selected from SEQ ID NOS: 12 to 14,

(b) a codon-optimized variant of (a) , or

(c) a transcribed RNA sequence of (a) or (b) .
The lipid nanoparticle composition of any one of claims 1 to 10, wherein the expression sequence comprises a coding sequence for the NeuroD1 peptide having the RNA sequence selected from SEQ ID NOS: 15 to 17.
The lipid nanoparticle composition of any one of claims 1 to 13, wherein the lipid nanoparticles comprises (a) a cationic lipid, (b) a steroid, (c) a phospholipid, and (d) a polymer conjugated lipid.
A linear precursor RNA molecule comprising a central region, wherein the central region comprises in the following order:

(a) a 3’ group I intron fragment or an analog thereof;

(b) an IRES;

(c) an expression sequence encoding one or more polypeptide comprising a NeuroD1 polypeptide, and

(d) a 5’ group I intron fragment or an analog thereof;

wherein the linear precursor RNA is capable of self-splicing into a circular RNA.
The linear precursor RNA molecule of claim 15, further comprising a pair of internal complementary sequences configured to form a second double stranded region by complementary base-pairing under a suitable hybridization condition, and wherein one of the pair of internal complementary sequences is located between elements (a) and (b) and the other of the pair of internal complementary sequences is located between elements (c) and (d) , respectively.
The linear precursor RNA molecule of claim 15 or 16, wherein the pair of internal complementary sequences have about 85%to about 100%complementarity when read in the opposite directions of one another.
The linear precursor RNA molecule of any one of claims 15 to 17, further comprising at least one internal spacer sequence located between elements (a) and (d) .
The linear precursor RNA molecule of claim 18, wherein the at least one internal spacer sequence comprises two internal spacer sequences located between elements (a) and (b) and between elements (c) and (d) , respectively.
The linear precursor RNA molecule of claim 19, wherein the internal spacer is about 10 to about 80 nucleotides long.
A circular RNA molecule formed by circulation of the linear precursor RNA molecule of any one of claims 15 to 20 through a ribozyme self-splicing reaction of the linear precursor RNA molecule.
A linear precursor RNA comprising the sequence of SEQ ID NO: 66 or 68.
A circular RNA molecule comprising the sequence of SEQ ID NO: 67 or 69.
A circular RNA molecule comprising, in the following order:

(a) a post-splicing 3’ group I intron fragment; optionally wherein post-splicing 3’ group I intron fragment comprises the sequence of SEQ ID NO: 64;

(b) an IRES;

(c) an expression sequence encoding one or more polypeptide comprising a NeuroD1 polypeptide, and

(d) a post-splicing 5’ group I intron fragment; optionally wherein post-splicing 5’ group I intron fragment comprises the sequence of SEQ ID NO: 65.
The circular RNA molecule of claim 24, further comprising at least one internal spacer sequence located between elements (a) and (d) .
The circular RNA molecule of claim 25, wherein the at least one internal spacer comprises two internal spacer sequences located between elements (a) and (b) and between elements (c) and (d) , respectively.
The circular RNA molecule of claim 25 and 26, wherein the internal spacer is about 10 to about 80 nucleotides long.
The circular RNA molecule of any one of claims 24 to 27 further comprising at least one pair of internal complementary sequences configured to form a double stranded region by complementary base-pairing under a suitable hybridization condition, wherein the at least one pair of internal complementary sequences are both located between elements (a) and (d) .
The circular RNA molecule of claim 28, wherein one of the pair of internal complementary sequences is located between elements (a) and (b) and the other of the pair of internal complementary sequences is located between elements (c) and (d) , respectively.
The circular RNA molecule of claim 28 or 29, wherein the pair of internal complementary sequences have about 85%to about100%complementarity when read in the opposite directions of one another.
The linear precursor RNA molecule or circular RNA molecule of any one of claims 15 to 30, wherein the expression sequence is a multi-cistronic sequence encoding the NeuroD1 polypeptide and at least one second polypeptide selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof.
The linear precursor RNA molecule or circular RNA molecule of claim 31, wherein the at least one second polypeptide comprises two polypeptides each independently selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof.
The linear precursor RNA molecule or circular RNA molecule of claim 31, wherein the expression sequence encodes:

(a) the NeuroD1 polypeptide;

(b) the NeuroD1 polypeptide and the Dlx2 polypeptide;

(c) the NeuroD1 polypeptide and the Isl1 polypeptide;

(d) the NeuroD1 polypeptide and the Ascl1 polypeptide;

(e) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Isl1 polypeptide;

(f) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ascl1 polypeptide;

(g) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ngn2 polypeptide;

(h) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ctip2 polypeptide;

(i) the NeuroD1 polypeptide, the Isl1 polypeptide, and the Ascl1 polypeptide;

(j) the NeuroD1 polypeptide, the Ils1 polypeptide, and the Lhx3 polypeptide;

(k) the NeuroD1 polypeptide, the Ascl1 polypeptide, and the Ctip2 polypeptide;

(l) the NeuroD1 polypeptide, the Dlx2 polypeptide, the Ascl1 polypeptide, and the Isl1 polypeptide; or

(m) the NeuroD1 polypeptide, the Dlx2 polypeptide, the Ascl1 polypeptide, and the Ctip2 polypeptide.
The linear precursor RNA molecule or circular RNA molecule of any one of claim 31 to 33, wherein the expression sequence comprises at least one ribosomal skipping element located between sequences encoding the NeuroD1 polypeptide and the at least one second polypeptide.
The linear precursor RNA molecule or circular RNA molecule of claim 34, wherein the ribosomal skipping element encodes a proteasome cleavage site selected from thosea-asigna virus 2A peptide (T2A) , porcine teschovirus-1 2 A peptide (P2A) , foot-and-mouth disease virus 2 A peptide (F2A) , equine rhinitis A vims 2A peptide (E2A) , cytoplasmic polyhedrosis vims 2A peptide (BmCPV 2A) , or flacherie vims of B. mori 2A peptide (BmIFV 2A) .
The linear precursor RNA molecule or circular RNA molecule of any one of claims 15 to 35, wherein the encoded NeuroD1 polypeptide is human NeuroD1 or a functional variant thereof; wherein optionally, the functional variant of human NeuroD1 comprises an amino acid sequence that at least about 90%, at least about 95%, at least about 97%, or at least about 99%sequence identity to SEQ ID NO: 1; wherein optionally, the functional variant of human NeuroD1 comprises an amino acid sequence that at least about 90%, at least about 95%, at least about 97%, or at least about 99%sequence identity to SEQ ID NO: 2.
The linear precursor RNA molecule or circular RNA molecule of claim 36, wherein the expression sequence comprises a coding sequence for the NeuroD1 polypeptide having:

(a) the DNA sequence selected from SEQ ID NOS: 12 to 14,

(b) a codon-optimized variant of (a) , or

(c) a transcribed RNA sequence of (a) or (b) .
The linear precursor RNA molecule or circular RNA molecule of claim 36, wherein the expression sequence comprises a coding sequence for the NeuroD1 peptide having the RNA sequence selected from SEQ ID NOS: 15 to 17.
A lipid nanoparticle composition comprising (a) cationic lipid, (b) a steroid, (c) a phospholipid,

(d) a polymer conjugated lipid, and (e) one or more of the linear precursor RNA molecule or circular RNA molecule of any one of claims 15 to 38.
A composition comprising a plurality of species of nucleic acid molecules each comprising an expression sequence, wherein each expression sequence encodes one or more polypeptide comprising a NeuroD1 polynucleotide, and wherein the one or more polypeptides encoded by at least two species of the plurality of species of nucleic acid molecules are different.
The composition of claim 40, wherein the one or more polypeptide encoded by at least one species of the plurality of species of nucleic acid molecules further comprises at least one second polypeptide selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof.
The composition of claim 40, wherein the one or more polypeptide encoded by at least one species of the plurality of species of nucleic acid molecules further comprises at least two second polypeptides selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof.
The composition of any one of claim 40 to 42, wherein the at least one species of the plurality of species of nucleic acid molecules comprises an ribosomal skipping element between the sequence encoding NeuroD1 and the sequence encoding the at least one second polypeptide selected from selected from Sox2, Dlx2, Isl1, Ascl1, Lhx3, Brn2, Ngn2, Gsx1, Tbr1, Ptf1a, Pax6, Otx2, Ctip2, Prox1, Nurr1, Myt1l, Brn3a, Lmx1a, and Lmx1b, or a functional derivative thereof.
The composition of claim 43, wherein the ribosomal skipping element encodes a cleavable fragment selected from T2A, P2A, F2A, E2A, BmCPV 2A and BmIFV 2A.
The composition of any one of claim 40 to 42, wherein one or more polypeptides encoded by the plurality of species of nucleic acid molecules comprise the polypeptide or combination of polypeptides selected from any one of (a) to (m) :

(a) the NeuroD1 polypeptide;

(b) the NeuroD1 polypeptide and the Dlx2 polypeptide;

(c) the NeuroD1 polypeptide and the Isl1 polypeptide;

(d) the NeuroD1 polypeptide and the Ascl1 polypeptide;

(e) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Isl1 polypeptide;

(f) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ascl1 polypeptide;

(g) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ngn2 polypeptide;

(h) the NeuroD1 polypeptide, the Dlx2 polypeptide, and the Ctip2 polypeptide;

(i) the NeuroD1 polypeptide, the Isl1 polypeptide, and the Ascl1 polypeptide;

(j) the NeuroD1 polypeptide, the Ils1 polypeptide, and the Lhx3 polypeptide;

(k) the NeuroD1 polypeptide, the Ascl1 polypeptide, and the Ctip2 polypeptide;

(l) the NeuroD1 polypeptide, the Dlx2 polypeptide, the Ascl1 polypeptide, and the Isl1 polypeptide; or

(m) the NeuroD1 polypeptide, the Dlx2 polypeptide, the Ascl1 polypeptide, and the Ctip2 polypeptide.
The composition of any one of claims 40 to 45, wherein the NeuroD1 is human NeuroD1 or a functional variant thereof; wherein optionally, the functional variant of human NeuroD1 comprises an amino acid sequence that has at least about 90%, at least about 95%, at least about 97%, or at least about 99%sequence identity to SEQ ID NO: 1; wherein optionally, the functional variant of human NeuroD1 comprises an amino acid sequence that has at least about 90%, at least about 95%, at least about 97%, or at least about 99%sequence identity to SEQ ID NO: 2.
The composition of any one of claims 40 to 45, wherein the expression sequence comprises a coding sequence for the NeuroD1 polypeptide having:

(a) the DNA sequence selected from SEQ ID NOS: 12 to 14,

(b) a codon-optimized variant of (a) , or

(c) a transcribed RNA sequence of (a) or (b) .
The composition of any one of claims 40 to 45, wherein the expression sequence comprises a coding sequence for the NeuroD1 peptide having the RNA sequence selected from SEQ ID NOS: 15 to 17.
The composition of any one of claims 40 to 48, wherein the plurality of species of nucleic acid molecules comprise at least one linear mRNA molecule.
The composition of claim 49, wherein the linear mRNA molecule further comprises a 3’-UTR and/or a 5’-UTR; optionally wherein the 5’-UTR comprises the sequence selected from the group consisting of SEQ ID NOS: 53 and 55; optionally wherein the 3’-UTR comprises the sequence selected from the group consisting of SEQ ID NOS: 57 and 59.
The composition of any one of claims 40 to 48, wherein the plurality of species of nucleic acid molecules comprise at least one circular RNA molecule.
The composition of claim 51, wherein the circular RNA molecule comprises, in the following order:

(a) a post-slicing 3’ group I intron fragment or an analog thereof; optionally the post-slicing 3’ group I intron fragment comprises the sequence of SEQ ID NO: 64;

(b) an IRES;

(c) an expression sequence encoding one or more polypeptide comprising a NeuroD1 polypeptide, and

(d) a post-slicing 5’ group I intron fragment or an analog thereof; optionally the post-slicing 5’ group I intron fragment comprises the sequence of SEQ ID NO: 65.
The composition of claim 52, wherein the circular RNA molecule further comprises at least one internal spacer sequence located between elements (a) and (d) .
The composition of claim 53, wherein the at least one internal spacer comprises two internal spacer sequences located between elements (a) and (b) and between elements (c) and (d) , respectively.
The composition of claim 53 or 54, wherein the internal spacer is about 10 to about 80 nucleotides long.
The composition of any one of claims 40 to 48, wherein the plurality of species of nucleic acid molecules comprise at least one linear precursor RNA molecule, wherein the linear precursor RNA molecule comprises a central region, wherein the central region comprises in the following order:

(a) a 3’ group I intron fragment or an analog thereof;

(b) an IRES;

(c) an expression sequence encoding one or more polypeptide comprising a NeuroD1 polypeptide, and

(d) a 5’ group I intron fragment or an analog thereof.
The composition of claim 56, wherein the linear precursor RNA molecule further comprises at least one internal spacer sequence located between elements (a) and (d) .
The composition of claim 57, wherein the at least one internal spacer sequence comprises two internal spacer sequences located between elements (a) and (b) and between elements (c) and (d) , respectively.
The composition of any one of claims 40 to 58, further comprising at least one lipid.
The composition of claim 59, wherein the at least one lipid forms lipid nanoparticles encompassing one or more species of the plurality of species of nucleic acid molecules.
The composition of claim 60, wherein the lipid nanoparticle comprises (a) a cationic lipid, (b) a steroid, (c) a phospholipid, and (d) a polymer conjugated lipid.
The composition of any one of claims 59 to 61, wherein the composition is a lipid nanoparticle composition.
A method of converting a starting population of glial cells into functional neurons, comprising contacting the starting population of glial cells with (a) the lipid nanoparticle composition of any one of claims 1 to 14, 39 or 62, (b) the linear precursor RNA molecule or the circular RNA molecule of any one of claims 15 to 38, and/or (c) the composition comprising the plurality of species of nucleic acid molecules of any one of claims 40 to 62 under a suitable condition, wherein upon the contacting, at least one glial cell in the starting population trans-differentiates into a functional neuron.
The method of claim 63, wherein the starting population of glial cells comprises astrocytes, NG2 cells and/or microglia cells.
The method of claim 63 or 64, wherein the functional neuron has at least one neuronal phenotype selected from neuronal morphology, expression of one or more neuronal marker, lack of expression of one or more glial cell marker, electrophysiologic characteristics of neurons, exon, dendrite and/or synapse formation, and release of neurotransmitters.
The method of claim 65, wherein the functional neuron expresses one or more neuronal markers selected from doublecortin (DCX) , class III beta tubulin (TUJ1) , neuronal specific nuclear protein (NeuN) , microtubule associated protein 2 (Map2) , RNA Binding Protein, MRNA Processing Factor (Rbpms) , brain-specific homeobox/POU domain protein 3A (Brn3a) , and Opsins.
The method of claim 65, wherein the trans-differentiated functional neuron stops expressing one or more glial cell marker selected from glia fibrillary acidic protein (GFAP) , aldehyde dehydrogenase 1 family, member L1 (AldhlL1) , S100 calcium-binding protein B (S100β) , SRY-box transcription factor 9 (Sox9) .
The method of claim 65, wherein the functional neuron is capable of firing action potentials.
The method of claim 65, wherein the functional neuron releases neurotransmitters selected from glutamate, GABA, dopamine, glycine, serotonin, and noradrenaline.
The method of any one of claims 63 to 69, wherein the starting population of glial cells are in an in vitro cell culture.
The method of any one of claims 63 to 69, wherein the starting population of glial cells are located in situ in a subject; optionally wherein the glial cells are located in the brain or spinal cord of the subject; optionally wherein the glial cells are located in the peripheral nervous system of the subject; optionally wherein the glial cells are located in the eye of the subject.
The method of claim 71, wherein the glial cells are located in the striatum of the brain.
The method of claim 72, wherein the contacting is performed by administering an effective amount of (a) the lipid nanoparticle composition of any one of claims 1 to 14, 39 or 62, (b) the linear precursor RNA molecule or the circular RNA molecule of any one of claims 15 to 38, and/or (c) the composition comprising the plurality of species of nucleic acid molecules of any one of claims 40 to 62 to the subject.
A method of producing a neuronal phenotype in a glial cell comprising contacting the glial cells with (a) the lipid nanoparticle composition of any one of claims 1 to 14, 39 or 62, (b) the linear precursor RNA molecule or the circular RNA molecule of any one of claims 15 to 38, and/or (c) the composition comprising the plurality of species of nucleic acid molecules of any one of claims 40 to 62 under a suitable condition, wherein upon the contacting, the glial cell produces a detectable neuronal phenotype.