WO2025080832A1 - Compositions and methods for replicon-mediated gene therapy - Google Patents

Abstract

Provided herein are methods and compositions related to the finding that specific mutations in nonstructural proteins of self-replicating RNA molecules including RNA replicons, such as Sindbis RNA replicons or Venezuelan equine encephalitis (VEE) replicons, can stabilize the longevity of the RNA replicon and can stabilize the expression of the cargo proteins encoded by the self-replicating RNA molecule or RNA replicon. Also disclosed herein are compositions and methods directed to the discovery that certain viral proteins, including viral proteins that repress internal cellular innate immunity, can stabilize the payload expression and replication of the replicons. Technologies provided herein provide stabilization mechanisms that can improve vaccines, gene therapies, and/or other gene delivery methods.

Description

COMPOSITIONS AND METHODS FOR REPLICON-MEDIATED GENE

THERAPY

[0001] This application claims priority of U.S. Provisional Application No. 63/543,330 filed October 10, 2023, which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on October 9, 2024, is named ARCD_P0819WO_Sequence_Listing.xml and is 95,810 bytes in size.

BACKGROUND

I. Field of the Invention

[0003] This invention relates to the field of genetic engineering, virology, and medicine.

II. Background

[0004] Replicons are self-amplifying recombinant RNA molecules expressing proteins sufficient for their own replication, but which do not produce infectious particles. Replicons can persist in cells and are passed on during cell division, enabling quick, efficient and high throughput testing of drug candidates that act on viral transcription, translation and replication. Safety of viral replicons has made them a popular tool for antiviral R&D and have been used to investigate a broad spectrum of antiviral compounds.

SUMMARY

[0005] The disclosure relates to the finding that specific mutations in nonstructural proteins of self-replicating RNA molecules including RNA replicons, such as Sindbis RNA replicons or Venezuelan equine encephalitis (VEE) replicons, can stabilize the longevity of the RNA replicon and can stabilize the expression of the cargo proteins, which can be referred to herein as proteins of interest, encoded by the self-replicating RNA molecule or RNA replicon. The disclosure also relates to the findings that including certain viral proteins, including viral proteins that repress internal cellular innate immunity, can stabilize the payload expression and replication of the replicons. Both stabilization mechanisms can improve vaccines, gene therapies, or other gene delivery methods. The mutations can be found by directed evolution, including by challenging cells with an increasing amount of a selection pressure, such as a cytotoxic agent (e.g. methotrexate), where the self-replicating RNA molecule or RNA replicon encodes a protein that protects a cell comprising the self-replicating RNA molecule or RNA replicon from the cytotoxic agent.

[0006] In various aspects, an RNA replicon comprising one mutation in nonstructural protein 3 (nPs3) of the RNA replicon and one mutation in nonstructural protein 4 (nPs4) of the RNA replicon is disclosed. The RNA replicon can be an RNA capable of self-replicating in a cell. The RNA replicon can be derived from a virus, such as an alphavirus. The RNA replicon can comprise segments of RNA from a virus. The RNA replicon can have mutations in various proteins, such as nPs3 an/ornPs4.. In various aspects, the mutation in nPs3 comprises an N252S mutation. In various aspects, the mutation in nPs4 comprises a T564A mutation. In various aspects, the RNA replicon is a Sindbis RNA replicon. In various aspects, the RNA replicon is derived from a Sindbis virus.

[0007] In various aspects, a self-replicating RNA molecule comprising one mutation in nonstructural protein 3 (nPs3) of the self-replicating RNA molecule and one mutation in nonstructural protein 4 (nPs4) of the self-replicating RNA molecule is disclosed. The selfreplicating RNA molecule can be derived from a virus, such as an alphavirus. The selfreplicating RNA molecule can comprise segments of RNA from a virus. The self-replicating RNA molecule can have mutations in various proteins, such as nPs3 an/or nPs4.. In various aspects, the mutation in nPs3 comprises an N252S mutation. In various aspects, the mutation in nPs4 comprises a T564A mutation. In various aspects, the self-replicating RNA molecule is a Sindbis RNA replicon. In various aspects, the self-replicating RNA molecule is derived from a Sindbis virus.

[0008] In various aspects, the RNA replicon and/or the self-replicating RNA comprise at least one viral protein, wherein the viral protein is capable of suppressing a cellular immune response. In certain aspects, the viral protein is hepatitis C nonstructural protein 3, hepatitis C nonstructural protein 4, hepatitis C nonstructural protein 5 A, Ebola virus VP35, Measles V antigen, Kaposi sarcoma associated herpesvirus ORF52, influenza H7N9 nonstructural protein 1, vaccinia Bl 8R, Dengue virus 3 nonstructural protein 5, herpes simplex virus- 1 infected cell protein 1, zika virus nonstructural protein 2 A, or a combination thereof.

[0009] In various aspects, the RNA replicon encodes a protein of interest. In various aspects, the protein of interest is a protein deficient in a genetic disease, a cancer, an autoimmune disease, an infection, and/or any other disease caused by or amplified by a deficient protein. In various aspects, the protein of interest is mutated and/or deleted in a genetic disease, a cancer, an autoimmune disease, an infection, and/or any other disease caused by or amplified by a deficient protein. The protein of interest can be a protein that is known to cause or amplify a disease, including any disease described herein. The protein of interest can be a protein that, when mutated, deleted, or otherwise having a loss of function, is known to cause or amplify a disease, including any disease described herein. In various aspects, the protein of interest is a steroid sulfatase (STS), Col7Al, an interleukin (such as IL- 10), a receptor (such as an IL-23 decoy receptor), or flagellin. In various aspects, the genetic disease is a recessive X- linked ichthyosis.

[0010] Also disclosed are viral vectors, gene-delivery vectors, gene-delivery platforms, therapeutic compositions, gene therapy products, or other compositions made by the human hand, any of which comprising one or more of the RNA replicons and/or self-replicating RNA disclosed herein. In some aspects, the composition comprises a nanoparticle, including any nanoparticle disclosed herein. In some aspects, the RNA replicon and/or self-replicating RNA are encapsulated in the nanoparticle. In some aspects, the nanoparticle comprises an ionizable lipid, a helper lipid, a sterol, and a DMG-PEG. In certain aspects, the ionizable lipid comprises D-Lin-MC3-DMA. In certain aspects, the helper lipid comprises (1,2-DSPC). In certain aspects, the sterol comprises cholesterol. In certain aspects, the DMG-PEG comprises DMG- PEG2000. In some aspects, the nanoparticle is capable of localizing in the liver of a patient. In some aspects, administration of a composition comprising the nanoparticle results in the localization of the nanoparticle and/or RNA replicon and/or self-replicating RNA in the liver of the patient.

[0011] Also disclosed are methods of using the RNA replicons, or other compositions, disclosed herein. The methods include methods of treating, methods of reducing the severity of a disease, methods of preventing a disease, methods of inhibiting disease progression, and/or methods of reversing the effects of a lack of a protein. The methods can comprise administering to the patient an effective amount of any of the RNA replicons disclosed herein, any of the expression constructs disclosed herein, any of the gene delivery platforms disclosed herein, and/or any of the therapeutic compositions disclosed herein. The patient can have, be suspected of having, or be diagnosed with having any of the diseases disclosed herein, including a recessive X-linked ichthyosis and dystrophic epidermolysis bullosa (including recessive dystrophic epidermolysis bullosa).

[0012] Also disclosed are methods of increasing expression of a protein of interest, methods of delivering a protein of interest to a cell of interest, methods of localizing protein of interest expression in a tissue, methods of localizing protein of interest expression on the skin of a patient, and methods of localizing protein of interest expression in the liver of a patient. The method can comprise contacting a cell of interest with a replicon or composition disclosed herein. The method can comprise administering a replicon or composition disclosed herein to a patient.

[0013] Certain aspects of the present disclosure are characterized through the following enumerated aspects.

[0014] Aspect 1 is an RNA replicon comprising one mutation in nonstructural protein 3 (nPs3) of the RNA replicon and one mutation in nonstructural protein 4 (nPs4) of the RNA replicon.

[0015] Aspect 2 is the RNA replicon of aspect 1, wherein the mutation in nPs3 comprises an N252S mutation.

[0016] Aspect 3 is the RNA replicon of aspect 1 or 2, wherein the mutation in nPs4 comprises a T564A mutation.

[0017] Aspect 4 is the RNA replicon of any one of aspects 1 to 3, wherein the RNA replicon is a Sindbis RNA replicon.

[0018] Aspect 5 is the RNA replicon of any one of aspects 1 to 4, wherein the RNA replicon encodes a protein of interest.

[0019] Aspect 6 is the RNA replicon of aspect 5, wherein the protein of interest is a protein deficient in a genetic disease, a cancer, an autoimmune disease, an infection, and/or any other disease caused by or amplified by a deficient protein.

[0020] Aspect 7 is the RNA replicon of aspect 5 or 6, wherein the protein of interest is mutated and/or deleted in a genetic disease, a cancer, an autoimmune disease, an infection, and/or any other disease caused by or amplified by a deficient protein.

[0021] Aspect 8 is the RNA replicon of any one of aspects 5 to 7, wherein the protein of interest is a steroid sulfatase (STS).

[0022] Aspect 9 is the RNA replicon of any one of aspects 6 to 8, wherein the genetic disease is a recessive X-linked ichthyosis.

[0023] Aspect 10 is the RNA replicon of any one of aspects 6 to 9, further comprising at least one viral protein, wherein the viral protein is capable of suppressing a cellular immune response.

[0024] Aspect 11 is the RNA replicon of aspect 10, wherein the viral protein is hepatitis C nonstructural protein 3, hepatitis C nonstructural protein 4, hepatitis C nonstructural protein 5A, Ebola virus VP35, Measles V antigen, Kaposi sarcoma associated herpesvirus ORF52, influenza H7N9 nonstructural protein 1, vaccinia Bl 8R, Dengue virus 3 nonstructural protein 5, herpes simplex virus- 1 infected cell protein 1, zika virus nonstructural protein 2A, or a combination thereof.

[0025] Aspect 12 is a self-replicating RNA molecule encoding nonstructural proteins comprising one mutation in nonstructural protein 3 (nPs3) of the self-replicating RNA molecule and one mutation in nonstructural protein 4 (nPs4) of the self-replicating RNA moelcule.

[0026] Aspect 13 is the self-replicating RNA molecule of aspect 12, wherein the mutation in nPs3 comprises an N252S mutation.

[0027] Aspect 14 is the self-replicating RNA molecule of aspect 12 or 13, wherein the mutation in nPs4 comprises a T564A mutation.

[0028] Aspect 15 is the self-replicating RNA molecule of any one of aspects 12 to 14, wherein the self-replicating RNA molecule is a Sindbis RNA replicon.

[0029] Aspect 16 is the self-replicating RNA molecule of any one of aspects 12 to 15, wherein the self-replicating RNA molecule encodes a protein of interest.

[0030] Aspect 17 is the self-replicating RNA molecule of any one of aspects 12 to 16, further comprising at least one viral protein, wherein the viral protein is capable of suppressing a cellular immune response.

[0031] Aspect 18 is the self-replicating RNA molecule of aspect 17, wherein the viral protein is hepatitis C nonstructural protein 3, hepatitis C nonstructural protein 4, hepatitis C nonstructural protein 5 A, Ebola virus VP35, Measles V antigen, Kaposi sarcoma associated herpesvirus ORF52, influenza H7N9 nonstructural protein 1, vaccinia B18R, Dengue virus 3 nonstructural protein 5, herpes simplex virus-1 infected cell protein 1, zika virus nonstructural protein 2A, or a combination thereof.

[0032] Aspect 19 is the self-replicating RNA molecule of aspect 16, wherein the protein of interest is a protein deficient in a genetic disease, a cancer, an autoimmune disease, an infection, and/or any other disease caused by or amplified by a deficient protein.

[0033] Aspect 20 is the self-replicating RNA molecule of aspect 16 or 19, wherein the protein of interest is mutated and/or deleted in a genetic disease, a cancer, an autoimmune disease, an infection, and/or any other disease caused by or amplified by a deficient protein.

[0034] Aspect 21 is the self-replicating RNA molecule of any one of aspects 16 to 20, wherein the protein of interest is a steroid sulfatase (STS).

[0035] Aspect 22 is the self-replicating RNA molecule of any one of aspects 19 to 21, wherein the genetic disease is a recessive X-linked ichthyosis. [0036] Aspect 23 is the RNA replicon encoding at least one viral protein, wherein the viral protein is capable of suppressing a cellular immune response.

[0037] Aspect 24 is the RNA replicon of aspect 23, wherein the viral protein is hepatitis C nonstructural protein 3, hepatitis C nonstructural protein 4, hepatitis C nonstructural protein 5A, Ebola virus VP35, Measles V antigen, Kaposi sarcoma associated herpesvirus ORF52, influenza H7N9 nonstructural protein 1, vaccinia Bl 8R, Dengue virus 3 nonstructural protein 5, herpes simplex virus- 1 infected cell protein 1, zika virus nonstructural protein 2A, or a combination thereof.

[0038] Aspect 25 is the RNA replicon of aspect 23 or 24, wherein the replicon is a Venezuelan equine encephalitis replicon.

[0039] Aspect 26 is the RNA replicon of aspect any one of aspects 23 to 25, wherein the replicon comprises the sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0040] Aspect 27 is the RNA replicon of aspect any one of aspects 18x1 to 18x3, wherein the replicon comprises a nucleic acid encoding a protein of interest.

[0041] Aspect 28 is the RNA replicon of aspect 27, wherein the protein of interest is COL7A1, a steroid sulfatase, IL- 10, an IL-23 decoy receptor, or filaggrin.

[0042] Aspect 29 is an RNA replicon comprising the nucleic acid of SEQ ID NO: 1.

[0043] Aspect 30 is an RNA replicon comprising the nucleic acid of SEQ ID NO:2.

[0044] Aspect 31 is an RNA replicon comprising the nucleic acid of SEQ ID NO:3.

[0045] Aspect 32 is an RNA replicon comprising the nucleic acid of SEQ ID NO:4.

[0046] Aspect 33 is an RNA replicon comprising the nucleic acid of SEQ ID NO:5.

[0047] Aspect 34 is an RNA replicon comprising the nucleic acid of SEQ ID NO:6.

[0048] Aspect 35 is an expression construct comprising the RNA replicon of any one of aspects 1 to 11 and/or 23 to 34 and/or the self-replicating RNA molecule of any one of aspects 12 to 22.

[0049] Aspect 336 is a vaccine comprising the RNA replicon of any one of aspects 1 to 11 and/or 23 to 34, the self-repli eating RNA molecule of any one of aspects 12 to 22, and/or the expression construct of aspect 36.

[0050] Aspect 37 is a gene delivery platform comprising the RNA replicon of any one of aspects 1 to 11 and/or 23 to 34, the self-replicating RNA molecule of any one of aspects 12 to 22, and/or the expression construct of aspect 35. [0051] Aspect 38 is a therapeutic composition comprising the RNA replicon of any one of aspects 1 to 11 and/or 23 to 34, the self-replicating RNA molecule of any one of aspects 12 to 22, and/or the expression construct of aspect 35.

[0052] Aspect 39 is the therapeutic composition of aspect 38, further comprising a nanoparticle.

[0053] Aspect 40 is the therapeutic composition of aspect 39, wherein the nanoparticle comprises an ionizable lipid, a helper lipid, a sterol, and a DMG-PEG.

[0054] Aspect 41 is the therapeutic composition of aspect 40, wherein the ionizable lipid comprises D-Lin-MC3-DMA, wherein the helper lipid comprises (1,2-DSPC), wherein the sterol comprises cholesterol, and/or wherein the DMG-PEG comprises DMG-PEG2000.

[0055] Aspect 42 is the therapeutic composition of any one of aspects 38 to 41, wherein the therapeutic composition is formulated for topical administration or intravenous administration.

[0056] Aspect 43 is a method of treating a disease in a patient, the method comprising administering to the patient an effective amount of the RNA replicon of any one of aspects 1 to 11 and/or 23 to 34, the self-replicating RNA molecule of any one of aspects 12 to 22, the expression construct of aspect 35, the vaccine of aspect 36, the gene delivery platform of aspect 37, and/or the therapeutic composition of any one of aspects 38 to 41.

[0057] Aspect 44 is the method of aspect 43, wherein the patient has, is suspected of having, or is diagnosed with having a deficiency and/or mutation in a specific protein.

[0058] Aspect 45 is the method of aspect 44, wherein the specific protein is a steroid sulfatase.

[0059] Aspect 46 is the method of aspect 43, wherein the RNA replicon encodes a protein of interest, and wherein the patient has, is suspected of having, or is diagnosed with having a deficiency and/or mutation in the protein of interest and/or the patient has, is suspected of having, or is diagnosed with having a disease associated with the protein of interest.

[0060] Aspect 47 is the method of aspect 46, wherein the protein of interest is a steroid sulfatase.

[0061] Aspect 48 is the method of any one of aspects 43 to 47, wherein the patient has, is suspected of having, or has been diagnosed with having a genetic disease, a cancer, an autoimmune disease, an infection, and/or any other disease caused by or amplified by a deficient protein.

[0062] Aspect 49 is the method of aspect 48, wherein the genetic disease, the cancer, the autoimmune disease, the infection is caused by or amplified by a deficient protein. [0063] Aspect 50 is the method of aspect 49, wherein the deficient protein comprises a mutated protein and/or deleted protein.

[0064] Aspect 51 is the method of any one of aspects 43 to 50, wherein the patient has, is suspected of having, or has been diagnosed with having a recessive X-linked ichthyosis.

[0065] Aspect 52 is the method of any one of aspects 43 to 50, wherein the patient has, is suspected of having, or has been diagnosed with having dystrophic epidermolysis bullosa.

[0066] Aspect 53 is a method of treating X-linked ichthyosis in a patient, the method comprising administering an RNA replicon encoding a steroid sulfatase and a viral protein to the patient.

[0067] Aspect 54 is a method of treating dystrophic epidermolysis bullosa in a patient, the method comprising administering an RNA replicon encoding Col7Al and a viral protein to the patient.

[0068] Aspect 55 is a method of increasing expression of a protein of interest in a cell of interest, the method comprising contacting the cell with the RNA replicon the RNA replicon of any one of aspects 1 to 11 and/or 23 to 33, the self-replicating RNA molecule of any one of aspects 12 to 22, the expression construct of aspect 35, the vaccine of aspect 36, the gene delivery platform of aspect 37, and/or the therapeutic composition of any one of aspects 38 to 41.

[0069] Aspect 56 is the method of aspect 55, wherein the cell of interest is a skin cell.

[0070] Aspect 57 is the method of aspect 55, wherein the cell of interest is a liver cell.

[0071] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0072] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Any term used in singular form also comprise plural form and vice versa.

[0073] As used herein, the terms “or” and “and/or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” “(x and z) or y,” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an aspect or aspect.

[0074] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0075] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0076] It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0077] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0078] Use of the one or more sequences or compositions may be employed based on any of the methods described herein. Other aspects and embodiments are discussed throughout this application. Any embodiment or aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa.

[0079] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the invention may apply to any other embodiment or aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application.

[0080] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0082] FIG. 1: Directed evolution for RNA replicon. To select novel replicon vector for persistent transgene expression, the inventors developed a recombinant Sindbis replicon vector that encodes tdTomato and DHFR (separated by a P2A self-cleavable peptide). Upon delivery to primary mouse keratinocytes, the cells were exposed to increasing concentration of MTX to force the evolution of RNA replicon that can increase expression of cargo genes. Mutations in RNA replicon were determined by Sanger Sequencing after directed evolution.

[0083] FIGs. 2A-2D Development of novel RNA replicon vector for somatic gene delivery. (A) Gradual increasing of methotrexate (MTX) concentration enforces Sindbis replicon evolution to achieve higher expression of DHFR, as indicated by enhanced coexpression of tdTomato. (B) Sanger sequencing of RNA replicon indicates the emergence of novel mutations in cells survived at high concentration of MTX. (C-D) Luciferase expression mediated by mRNA or different RNA replicons was determined by bioluminescence imaging (C) and quantified over time upon intradermal delivery (D). n=3, error bars represent standard deviation.

[0084] FIGs. 3A-3E: Development of STS KO mouse model. (A) Design of STS- targeting gRNA (green box), which recognize the sequence at exon 2 (grey box) of mouse STS gene. (B) Typical PCR genotyping of STS KO animals. CRISPR targeting leads to an -200 bp deletion at exon 2. (C) STS KO mice were bom at expected Mendelian ratio but appeared smaller. (D) Immunoblot (IB) analysis of primary keratinocytes isolated from WT or KO skin for STS expression. (E) Skin histology or WT or STS KO animals (P2) shows hyperkeratosis upon loss of STS.

[0085] FIG. 4 : Deletion of STS in human cells was achieved by lentiviral vector encoding cas9 and AViS'-gRNA.

[0086] FIG 5: Generation of reporter VEE replicon vector. Schematic showing generation of VEE-NLuc replicon vector with co-expression of different viral genes that can suppress host innate immune response. [0087] FIG 6: Live luminescence imaging of NLuc mRNA (control), WT VEE-NLuc, and VEE-NLuc-HCV NS3/4 injection in nude mice. Each nude mice were intradermally injected with 2 doses of mRNA NLuc control or VEE-NLuc or VEE-NLuc-HCV NS3/4 (3 ug) in both of their lower limbs (each side with the same amount of mRNA). At 2-, or 30-days post-injection (dpi), NLuc substrates FFZ were intraperitoneally administrated, and the bioluminescence signal was obtained using an in vivo imaging system (IVIS).

[0088] FIG 7: Live luminescence imaging of NLuc mRNA or different VEE replicon injection in vivo. Each mice were intradermally injected with 2 dose of mRNA control or VEE replicon encoding different viral anti-immunity genes (3 ug) (Kaposi sarcoma herpesvirus ORF52, Zika NS2A, HCV NS5A) in lower limbs. At 2 or 30-days post-injection (dpi), NLuc substrates FFZ were intraperitoneally administrated, and the bioluminescence signal was obtained using an in vivo imaging system (IVIS).

[0089] FIG 8: Live luminescence imaging of NLuc mRNA or different VEE replicon intravenous injection in vivo. Each mice were intravenously injected with 1 dose of mRNA NLuc control or VEE-NLuc or VEE-NLuc-HCV NS3/4 (3 ug). At 7 days post-injection (dpi), NLuc substrates FFZ were intraperitoneally administrated, and the bioluminescence signal in liver was obtained using an in vivo imaging system (IVIS).

[0090] FIGs. 9A-9E: Expression of different GOI (gene of interest) in vitro with replicon vectors. Primary keratinocytes were transfected with mRNA or VEE-WT or VEE- HCV NS3/4 (3 ug) encoding different cargo genes. At different time points (1, 7, and 15 days post-transfection), expression of different genes were quantified by real time PCR. Note mRNA-mediated expression decays rapidly, and becomes non-detectible within 7 days. VEE replicon vector with HCV NS3/4 can greatly enhance the expression durability for different cargo genes.

[0091] FIG. 10: RDEB results from lack of functional COL7A1 which inhibits anchoring fibril formation in the epidermal membrane basement zone and results in blistering.

[0092] FIG. 11: A topical delivery of a dual-vector, self-amplifying RNA replicon, which will enable long-term expression of COL7A1 to treat RDEB.

DETAILED DESCRIPTION

[0093] Certain aspects of the disclosure relate to engineered replicons. In some aspects, the replicon has one or more mutations in the replicon sequence that are capable of increasing expression and/or replication of the replicon. In some aspects, the replicon is a Venezuelan equine encephalitis (VEE) replicon. In some aspects, the replicon derived from a Venezuelan equine encephalitis (VEE) replicon. In some aspects, the replicon is a Sindbis virus replicon. In some aspects, the replicon is derived from a Sindbis virus replicon. The replicons can include the replicons of SEQ ID NOs: 1-6. SEQ ID NO: 1 includes a VEE-NS3/4 empty vector, wherein a nucleic acid encoding a protein of interest (including any protein of interest disclosed herein) and/or viral protein (including any viral protein disclosed herein) can be inserted into the vector. In some aspects, the protein of interest and the viral protein are separated by a 2A sequence, such as a P2A sequence or T2A sequence. In some aspects, the protein of interest and the viral protein of interest are separated by an IRES sequence. In some aspects, the nucleic acid encoding the protein of interest and the nucleic acid encoding the viral protein are separated by nucleic acid sequence encoding a 2A sequence, such as a P2A sequence or T2A sequence. In some aspects, the nucleic acid encoding the protein of interest and the nucleic acid encoding the viral protein of interest are separated by an IRES sequence.

[0094] SEQ ID NO:2 includes a VEE-NS3/4 replicon comprising a filaggrin gene. SEQ ID NO:3 includes a VEE-NS3/4 replicon comprising a Col7Al gene. SEQ ID NO:4 includes a VEE-NS3/4 replicon comprising an STS gene. SEQ ID NO:5 includes a VEE-NS3/4 replicon comprising an IL-23 decoy receptor gene. SEQ ID NO:6 includes a VEE-NS3/4 replicon comprising an IL- 10 gene.

[0095] In some aspects, the protein of interest is inserted into SEQ ID NO: 1 before SEQ ID NO:7. In some aspects, the protein of interest is inserted into SEQ ID NO: 1 before SEQ ID NO:7.

GGCTGCGTGGTGATCGTGGGCAGGATCGTGCTGAGCGGCAGCGGCACCAGCGCGCCCATCAC GGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGG ACAAAAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACCTTCCTGGCA ACGTGCATCAATGGGGTATGCTGGACAGTCTACCACGGGGCCGGAACGAGGACCATCGCATC ACCCAAGGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGCTGGCCCG CTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGGTC ACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTC GCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGAC ACGCCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTT ATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACTCCTCTCC ACCAGCATGAtaattaattaaGAATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCA TGCCGCCTTAAAATTTTTATTTTATTTTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAA T AT T T C AAAAAAAAAAAAAAAAAAAAAAAAAAC G C G T C GAG G G GAAT T AAT T C T T GAAGAC G AAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTA AAT AC AT T C AAAT AT GTATCCGCTCAT GAGAC AAT AAC C C T GAT AAAT G C T T GAAT AAT AT T GAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCA TTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCA GTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTT TTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTA TTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGA CTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAAT TATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATC GGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGA TCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTG TAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGG CAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCT TCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCA TTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGT CAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCA T T G G T AAC T G T C AGAC C AAG TTTACTCATATATACTT T AGAT T GAT T T AAAAC TTCATTTTT AAT T T AAAAG GAT C T AG G T GAAGAT C C T T T T T GAT AAT C T C AT GAG C AAAAT C C C T T AAC G T GAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCC TTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTT GTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAG ATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGC

ACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGT CGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGA ACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCT ACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGG TAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTAT CTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTC AGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGAGCTCTAATACGACTCACTATAG

( SEQ ID NO : 7 )

[0096] The protein of interest may be any protein disclosed herein. In some aspects, the protein of interest is a protein that is mutated or absent in a disease. In some aspects, the protein of interest is a protein associated with a disease. In certain aspects, a patient described herein lacks the protein of interest.

I. Proteins

[0097] As used herein, a “protein” or “polypeptide” refers to a molecule comprising at least five amino acid residues. As used herein, the term “wild-type” refers to the endogenous version of a molecule that occurs naturally in an organism. In some embodiments, wild-type versions of a protein or polypeptide are employed, however, in many embodiments of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some embodiments, a modified/variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified/variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity. [0098] Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein or, optionally, a protein in which any signal sequence has been removed. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA/exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In particular embodiments, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide. The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

A. Variant Polypeptides

[0099] The following is a discussion of changing the amino acid subunits of a protein to create an equivalent, or even improved, second-generation variant polypeptide or peptide. For example, certain amino acids may be substituted for other amino acids in a protein or polypeptide sequence with or without appreciable loss of biological activity. Since it is the interactive capacity and nature of a protein that defines that protein’s functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and nevertheless produce a protein with similar or desirable properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes which encode proteins without appreciable loss of their biological utility or activity.

[0100] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.

[0101] Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the disclosure may affect 1, 2, 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, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to wild-type. A variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein. A variant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids. [0102] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.

[0103] Deletion variants typically lack one or more residues of the native or wild type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein.

[0104] Insertional mutants typically involve the addition of amino acid residues at a nonterminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.

[0105] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties. “Conservative amino acid substitutions” may involve exchange of a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.

[0106] Alternatively, substitutions may be “non-conservative”, such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.

1. Considerations for Substitutions

[0107] One skilled in the art can determine suitable variants of polypeptides as set forth herein using well-known techniques. One skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. The skilled artisan will also be able to identify amino acid residues and portions of the molecules that are conserved among similar proteins or polypeptides. In further embodiments, areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without significantly altering the biological activity or without adversely affecting the protein or polypeptide structure.

[0108] In making such changes, the hydropathy index of amino acids may be considered. The hydropathy profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (—0.4); threonine (—0.7); serine (—0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathy amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157: 105-131 (1982)). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein or polypeptide, which in turn defines the interaction of the protein or polypeptide with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and others. It is also known that certain amino acids may be substituted for other amino acids having a similar hydropathy index or score, and still retain a similar biological activity. In making changes based upon the hydropathy index, in certain embodiments, the substitution of amino acids whose hydropathy indices are within ±2 is included. In some aspects of the present disclosure, those that are within ±1 are included, and in other aspects of the present disclosure, those within ±0.5 are included. [0109] It also is understood in the art that the substitution of like amino acids can be effectively made based on hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigen binding, that is, as a biological property of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (—0.4); proline (-0.5+1); alanine (^_0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 are included, in other embodiments, those which are within ±1 are included, and in still other embodiments, those within ±0.5 are included. In some instances, one may also identify epitopes from primary amino acid sequences based on hydrophilicity. These regions are also referred to as “epitopic core regions.” It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0110] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides or proteins that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[OHl] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar proteins or polypeptides. One skilled in the art may choose not to make changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using standard assays for binding and/or activity, thus yielding information gathered from such routine experiments, which may allow one skilled in the art to determine the amino acid positions where further substitutions should be avoided either alone or in combination with other mutations. Various tools available to determine secondary structure can be found on the world wide web at expasy.org/proteomics/protein_structure.

[0112] In some embodiments of the disclosure, amino acid substitutions are made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinities, and/or (5) confer or modify other physicochemical or functional properties on such polypeptides. For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally occurring sequence. In such embodiments, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein or polypeptide.

II. Nucleic Acids

[0113] The term “polynucleotide” refers to a nucleic acid molecule that either is recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or noncoding sequences may, but need not, be present within a polynucleotide.

[0114] In this respect, the term “gene,” “polynucleotide,” or “nucleic acid” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein.

[0115] In certain embodiments, there are polynucleotide variants having substantial identity to the sequences and/or genes disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90%, preferably 95% and above, identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.

[0116] The nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. The nucleic acids can be any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and/or can comprise one or more additional sequences, for example, regulatory sequences, and/or be a part of a larger nucleic acid, for example, a vector. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.

A. Mutation

[0117] Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide that it encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more particular amino acid residues are changed using, for example, a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are changed using, for example, a random mutagenesis protocol. However it is made, a mutant polypeptide can be expressed and screened for a desired property.

[0118] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively changes the biological activity of a polypeptide that it encodes. See, eg., Romain Studer et al., Biochem. J. 449:581-594 (2013). For example, the mutation can quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity.

III. Obtaining Encoded Polypeptide Embodiments

A. Vectors

[0119] In some aspects, contemplated are expression vectors comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof. Expression vectors comprising the nucleic acid molecules may encode a protein of interest, including any protein described herein. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

B. Expression Systems

[0120] Numerous expression systems exist that comprise at least a part or all of the expression vectors discussed above. Prokaryote- and/or eukaryote-based systems can be employed for use with an embodiment to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Commercially and widely available systems include in but are not limited to bacterial, mammalian, yeast, and insect cell systems. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide using an appropriate expression system.

IV. Methods of Gene Transfer

[0121] Suitable methods for nucleic acid delivery to effect expression of compositions are anticipated to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Patents 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium mediated transformation (U.S. Patents 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation/inhibition mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

A. Host Cells

[0122] In another aspect, contemplated are the use of host cells into which a recombinant expression vector has been introduced. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. In certain aspects, the expression construct can be placed under control of a promoter that is linked to T-cell activation, such as one that is controlled by NFAT-1 or NF- KB, both of which are transcription factors that can be activated upon T-cell activation. One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

[0123] For stable transfection of mammalian cells, it is known, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods known in the arts.

B. Isolation

[0124] Methods of isolating RNA and RNA replicons are well known in the art.

V. Administration of Therapeutic Compositions

[0125] Certain aspects herein relate to the administration of a replicon disclosed herein or composition comprising a replicon disclosed herein. In some aspects, the administration results in the increased expression of a protein of interest.

[0126] Embodiments of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed.

[0127] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0128] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0129] In some embodiments, the therapy comprises an RNA replicon, a nucleic acid encoding for the RNA replicon protein, a vector comprising the nucleic acid encoding for the RNA replicon protein, or a cell comprising the RNA replicon. In some embodiments, a single dose of the therapy is administered. In some embodiments, multiple doses of the therapy are administered. In some embodiments, the therapy is administered at a dose of between 1 pg/kg and 5000 mg/kg. In some embodiments, the therapy is administered at a dose of at least, at most, or about 1, 2, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109_: 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128_: 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147^ 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185_: 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204^ 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223^ 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242^ 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 , 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 ng, mg, Pg/kg, or mg/kg.

[0130] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg/kg to 200 mg/kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg/kg, mg/kg, pg/day, or mg/day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.

[0131] In certain embodiments, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM. In another embodiment, the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein). In other embodiments, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 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, 50, 51, 52, 53,

54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,

79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 pM or any range derivable therein. In certain embodiments, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0132] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0133] It will be understood by those skilled in the art and made aware that dosage units of pg/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of pg/ml or mM (blood levels). It is also understood that uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0134] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 week intervals, including all ranges there between. [0135] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0136] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0137] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0138] The proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0139] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0140] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0141] Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0142] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above..

A. Pharmaceutical Compositions

[0143] In certain aspects, the compositions or agents for use in the methods, such as an RNA replicon, an expression construct, a vaccine, a gene therapy platform, and/or therapeutic composition, are suitably contained in a pharmaceutically acceptable carrier. The carrier is nontoxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the disclosure may be formulated into preparations for local delivery (i.e. to a specific location of the body, such as the skin or other tissue) or systemic delivery, in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration. Certain aspects of the disclosure also contemplate local administration of the compositions by coating medical devices and the like.

[0144] Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0145] The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.

[0146] In certain aspects, the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0147] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0148] In certain aspects, the pharmaceutical compositions are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable or solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[0149] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antgifungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.

[0150] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0151] In further aspects, the pharmaceutical compositions may include classic pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For treatment of conditions of the lungs, aerosol delivery can be used. Volume of the aerosol may be between about 0.01 ml and 0.5 ml, for example.

[0152] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[0153] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.

Examples

[0154] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Research Strategy

[0155] Genetic disorders are caused by variants affecting nuclear or mitochondria genes, combinations of variant genes and environmental factors, or alterations in the number or structure of one or more chromosomes or chromosome regions. Although individual inborn disorders are usually rare, they collectively represent a common health issue with significant socio-economic burden¹'³. Skin provides an essential barrier to protect us from various environmental damages. Skin ichthyoses are a group of heterogeneous genetic diseases that are characterized by hyperkeratosis, localized or generalized scaling, and often associated with xerosis, hypohydrosis, erythroderma, and recurrent infections⁴'⁷. So far, mutations in more than 50 genes have been shown to cause ichthyosis, which affect a variety of different cellular processes, ranging from DNA repair, lipid biosynthesis, cell adhesion, and skin differentiation. Recessive X-linked ichthyosis (RXLI) is the second most common form of inherited ichthyosis, with a prevalence of 1 :2000 to 1 :6000 males⁸'¹⁰. RXLI is caused by mutations in the STS gene on the X chromosome, which encodes microsomal steroid sulfatase. As a genetic disorder, RXLI is a life-long condition that is associated with multiple comorbidities and substantially diminishes patients’ quality of life. More effective treatment beyond current symptomatic management is urgently needed.

[0156] Somatic gene therapy provides a promising therapeutic approach for a variety of otherwise terminal or severely disabling genetic diseases¹¹. Although viral-based gene therapy has been extensively studied, use of viral vectors is associated with host immune response and potential insertional mutagenesis, limiting its efficacy and safety for clinical applications^12,13. In addition, viral approach is costly due to the complexity of viral vector manufacturing and the potential need for personalized treatment. RNA-based therapeutics have gained significant attention as an alternative approach to conventional gene therapy¹⁴'¹⁶. The RNA vaccines for COVID-19 have been a remarkable success^17,18, highlighting the key advantages of RNA vectors, such as fast development and production, low cost, high potency for protein production, and reduced risk for genotoxicity associated with viral vectors. However, RNA- based therapeutics also face significant technical hurdles for their clinical application in gene therapy because RNA molecules have a short half-life in vivo, limiting their therapeutic efficacy for genetic diseases. Self-amplifying RNA vectors (saRNAs) have been developed as a promising solution to the limitations of RNA-based therapeutics¹⁹'²¹. Unlike traditional RNA molecules, saRNAs are designed to contain a replicon derived from RNA virus, such as alphaviruses, which encodes viral nonstructural proteins as RNA replicase to amplify the RNA message in cells. By deletion of essential structural genes, RNA replicons are disabled viral vectors that are unable to revert to virulent. However, although the amplification mechanism allows for more persistent protein production, saRNA-driven expression still fades gradually in vitro and in vivo due to the cytopathic effect of the vector and innate immune response from the host cells²²'²⁴.

[0157] To circumvent these obstacles, the inventors have designed a continuous directed evolution platform (Fig. 1) to develop and select novel replicon vectors that can achieve durable and high expression within primary skin keratinocytes with intact innate immune responses. With this innovative system, the inventors have evolved the Sindbis virus-based replicon encoding dihydrofolate reductase (DHFR) in vitro by increasing concentration of methotrexate (MTX) ^25>26. The inventors identified two unique mutations located at the nonstructural protein 3 (nsPs3) and 4 (nsPs4), which can greatly enhance expression of the “cargo” gene under the subgenomic promoter in vitro and in vivo. In this exploratory proposal, as a proof of concept the inventors will examine the intriguing possibility that engineered replicon vector can serve as a novel platform for gene therapy of skin genetic disorders. Specifically, the inventors plan to further optimize the replicon vector for STS expression in both primary mouse keratinocytes and human skin organoid cultures, the inventors will explore the underlying molecular mechanisms whereby the two point mutations enhance the expression of cargo genes. To evaluate the therapeutic efficacy in a preclinical setting, the inventors have established an STS knockout (KO) strain with CRISPR (clustered regularly interspaced short palindromic repeats) approach²⁷. With this model, the inventors will determine replicon- mediated expression of STS in vivo. The proposed research will define a novel paradigm for RXLI treatment with an RNA replicon engineering approach.

[0158] Innovation:

[0159] Limit of current practice: As a genetic disease, most RXLI patients only receive symptomatic treatments⁸. In severe forms of RXLI, patients may benefit from topical keratolytics or steroid treatment, but these therapies are associated with strong side effects or toxicity⁸. Although traditional cutaneous gene delivery approach may provide an effective way to correct the mutation, this approach is costly due to the requirement for personalized treatment and the complexity of viral vector and cell manufacturing^28,29. Viral vector-based gene delivery is also associated with the risk of insertional mutagenesis and host immune response^12,13. Herpes simplex virus (HSV-l)-mediated gene delivery has been approved for treatment of dystrophic epidermolysis bullosa (DEB) ^30,31. However, expression mediated by HSV-1 is transient and manufacturing of the HSV-1 vector at high titer remains challenging^32,33. The cost for the treatment is estimated to be ~ $600,000 per year.

[0160] Although RNA therapeutics have^shown promise as vaccine for infectious diseases, its potential applicability for gene therapy has been limited by the short half-life of RNA molecules upon deliver In this proposal, the inventors will leverage the selfamplification mechanism of RNA replicon and the unique directed evolution platform to develop novel RNA vectors for long term and high expression of target genes in skin cells, the inventors will perform a proof-of-concept study to explore the possibility of RXLI treatment with engineered RNA replicon for corrective expression of STS in skin keratinocytes. The inventors will determine the therapeutic efficacy with both RXLI murine model recently developed by us and human 3D skin organotypic model. Our results will establish the key groundwork toward paradigm-shifting treatment for skin genetic disorders.

[0161] Approach:

[0162] Sindbis virus-based vector has been developed for heterologous gene expression in vitro²⁰’²¹. Sindbis virus-based vector can lead to high level expression of proteins up to 10⁸ molecules per cell because the non- structural genes can function as RNA replicase that can amplify the RNA in the cytoplasm (RNA replicon). However, its application has been limited to short-term transient expression due to the innate immune reaction and cytopathic effects induced by replicon vector amplification²²’²³. Previous studies have identified P726L or P726S mutation in the nsP2 (nonstructural protein 2) gene that can reduce cytopathic effects in different cell types and lead to long term expression of exogenous genes, but the mutation also dramatically inhibits RNA replication and target gene expression⁵⁵'⁵⁸. Most studies with Sindbis replicon used BHK-21 cells as a model that is deficient in interferon signaling. Persistent expression via replicon in primary cells with intact innate immune reaction, such as keratinocytes, remains challenging.

[0163] To develop a more effective RNA replicon vector for skin genetic disease, the inventors designed a directed evolution platform with wildtype (WT) Sindbis replicon (Fig. 1). Primary skin keratinocytes can withstand long-term culture in vitro without changing cell identity, making it an ideal host for replicon evolution. The process was initiated by transfection of primary mouse keratinocytes (DHFR KO) with a DNA vector encoding the Sindbis replicon with a CMV promoter. The recombinant replicon harbors the coding sequence for both a fluorescence reporter (tdTomato) and a selection marker DHFR for directed evolution²⁶. DHFR catalyzes the conversion of folate to tetrahydrofolate, which is required for purine, amino acid, and nucleoside biosynthesis. The folic acid analog MTX binds and inhibits DHFR, leading to cell death. Thus, surviving populations of cells exposed to sequentially increasing concentrations of MTX (0-10 pM) must contain increased levels of DHFR expression from the evolved replicon vectors (Fig. 1), as indicated by the enhanced co-expression of tdTomato (Fig. 2A).

[0164] To determine the potential changes on Sindbis replicon, the inevntors isolated total RNA from cells at different stages during evolution and sequenced the replicon after reversetranscription and cloning to a plasmid vector. For each stage, the inventors performed Sanger sequencing for more than 50 individual clones. Consistent with previous reports, the inventors identified the same non-cytopathic P726S mutation in nsP2, which became the predominant replicon specie at the initial stage of evolution (Fig. 2B). Interestingly, RNA replicons retrieved from cells at the final stage of evolution (10 pM MTX) contains a distinct specie, which harbors two point mutations at nsP3 (N252S) and nsP4 (T564A) (Fig. 2B). This novel replicon RNA appears to co-exist with the P726S mutant in keratinocytes survived with highest concentration of MTX, suggesting that the new mutant replicon can act in trans with the non-cytopathic RNA replicon to enhance expression of cargo genes without increasing cytotoxicity. To examine replicon-mediated expression in vivo, the inventors prepared different Sindbis vectors encoding luciferase by in vitro transcription. Upon intradermal injection with MC3 LNP, the inventors monitored luciferase expression by bioluminescence imaging. Consistent with expression in vitro, delivery of luciferase mRNA or WT Sindbis replicon led to significant but transient expression in skin (Fig. 2C and D). P726S mutation diminished expression in vivo, whereas the new N252S/T564A mutant alone cannot express luciferase. However, a mixture of P726S and N252S/T564A mutants (3: 1) can mediate markedly enhanced and durable expression of luciferase in skin. Bioluminescence signal remains strong 5 weeks post intradermal delivery (Fig. 2C and D). In this proposal, the inventors will examine the possibility for treatment of skin genetic diseases with this novel RNA replicon.

[0165] Specific Aim 1: Engineer RNA replicon vector to restore STS expression in keratinocytes.

[0166] RXLI is the second most common form of inherited ichthyosis, caused by mutations in the STS gene ^8,1°. The skin abnormalities of RXLI is caused by the impact of excess cholesterol sulfate, which affects lipid synthesis, organization of the lamellar lipids that provides the skin permeability barrier, comeodesmosome proteolysis, and epidermal differentiation. In this aim, the inventors will use engineered RNA replicon vector to rescue expression of STS in skin keratinocytes as a novel gene therapy for RXLI.

[0167] AimlA. Develop Sindbis RNA replicon vector for STS expression. Optimize Sindbis RNA replicon vector: Although the new replicon vector drives high and durable expression of cargo gene upon intradermal delivery, the inventors will continue the directed evolution process to determine whether the vector can be further optimized to mediate persistent expression in vitro and in vivo. The inventors will gradually increase MTX selection to 50 pM (MTX has extremely high toxicity beyond this concentration). Survived cells will be expanded, and RNA replicon sequence will be determined by Sanger sequencing upon reverse transcription and molecular cloning. At least 50 individual clones will be selected, and all 4 nonstructural protein encoding sequences will be fully sequenced, as described above. Efficacy of potential new mutations will be determined by RNA replicon delivery to primary keratinocytes in vitro and intradermal injection to skin in vivo. [0168] Prepare recombinant RNA replicon vector for STS expression: With standard molecular cloning, the inventors will engineer RNA replicon vectors with human STS gene inserted behind the subgenomic promoter. The inventors will clone STS to WT, P726S, and N252S/T564A replicon vectors. RNA replicon will be prepared by in vitro transcription, and expression of STS will be verified by immunoblots upon LNP delivery to primary mouse keratinocytes. Enzyme activity will be determined by sulfatase activity colorimetric kit in vitro (Sigma MAK276).

[0169] Statistical analysis: For each sample, the inventors will have at least three biological repeats. Statistical significance will be calculated with Prism software (GraphPad Inc.). For direct comparison between two groups, Student’s t-test will be used. For comparison among three or more groups, the inventors will perform one-way ANOVA analysis. P < 0.05 will be considered as statistically significant.

[0170] Potential outcomes: The inventors expect to develop recombinant RNA replicon vector for STS expression. Given our preliminary results, the inventors do not anticipate significant technical difficulty to carry out this aim. Although unlikely, if over-expression of STS leads to cytotoxicity, the inventors will incorporate synthetic regulatory elements in the replicon vector to modulate gene expression⁶². Aim IB. Determine the therapeutic efficacy in vivo with STS KO model. Lack of murine models represents a significant technical hurdle to develop novel treatment for RXLI. To address this issue, the inventors have generated STS KO mouse strain by CRISPR targeting approach. The guide RNA (gRNA) was selected to target the second exon of mouse STS gene at X chromosome, which localize at the 5’ end of the open reading frame (Fig. 3A). CRISPR-mediated targeting led to efficient generation of STS KO alleles, as determined by PCR genotyping and sequencing of the genomic sequence from the offspring (Fig. 3B). Neonatal mice genotypic for STS KO (male) were bom in the expected Mendelian numbers and can grow to adulthood, but usually appeared smaller than their WT littermates (Fig. 3C). Immunoblot analyses confirms the loss of STS expression in primary keratinocytes isolated from KO skin (Fig. 3D). Histological analysis indicates hyperkeratosis, the thickening of the stratum comeum in the KO skin, as expected (Fig. 3E). Successful development of the STS KO model offers us the unique platform to evaluate the therapeutic efficacy of RNA replicons in vivo.

[0171] Intradermal delivery of RNA replicon for RXLI treatment: RNA replicon encoding STS will be prepared by in vitro transcription. For intradermal delivery, the inventors will prepare MC3 LNP, which is composed of ionizable lipid DLin-MC3-DMA, helper lipid (1,2- DSPC), cholesterol and DMG-PEG2000 with a molar ratio of 50: 10:38.5: 1.5. The lipid mixture is dissolved in ethanol and RNA is dissolved in 50mM sodium acetate buffer (pH 5.0). 5 pg of RNA (STS mRNA, WT replicon, P726S replicon, N252S/T564A replicon, or mixture of P726S and N252S/T564A at 1 :3 ratio) will be delivered to adult KO mice (male, 12 weeks old) intradermally at ear skin.

[0172] Treated animals will be euthanized at different timepoints (0, 3, 7 days, 2, and 4 weeks). For histopathological analysis, the skin samples will be fixed in 10% formalin and embedded in paraffin. Expression of STS will be determined by IHC. H&E analysis will be used to quantify potential hyperkeratosis in skin. IHC analysis with antibodies for Ki-67, Keratin 14, Keratin 10, and Transglutaminase 1 (TGM1) will be used to detect cell proliferation and epidermal differentiation. TUNEL staining will be used to detect apoptotic cell death. All histological and IHC data will be analyzed and scored in blinded studies through the UChicago Pathology Core.

[0173] Skin barrier function measurements: TEWL (trans-epidermal water loss) measures the amount of water vapor evaporating from the surface of the skin, considered to be the standard procedure for assessing the skin barrier function. TEWL at treated skin will be determined at Northwestern SBDRC.

[0174] Statistical analysis: the inventors will use -150 mice for this aim. The number of mice per cohort (5 per group) was determined based on the power calculations in the program G’ Power 3.1.3 assuming at least 90% power and an effect size of .5 at the p-value threshold of 0.05, and verified using power calculator designed by Dr. Lenth R. These numbers will allow statistical significance required by the proposed studies.

[0175] Potential outcomes and caveats: Given our preliminary results, the inventors expect that WT replicon or STS mRNA can restore expression transiently, whereas the new mutant (mixture of P726S and N252S/T564A) can drive high level and persistent expression of STS in skin. Retroviral-based expression of STS can rescue skin abnormalities ex vivo with RXLI patient-derived keratinocytes⁶³. Thus, the inventors expect that STS expression with RNA replicon can restore normal skin architecture and barrier function in vivo.

[0176] The inventors used the same MC3 LNP for intradermal delivery of Luciferase- encoding replicon, the inventors anticipate similar efficacy for STS RNA delivery. However, pending initial results, the inventors are also interested to develop novel formulation of the LNP to include keratinocyte-targeting peptide and optimize the surface charge and lipid composition, aiming toward keratinocyte-specific gene delivery and potential topical application of the RNA/LNP for skin disease treatment ⁶⁴ This aim will be pursued in collaboration with Dr. Jun Huang (co-I), who has extensive expertise in nanoparticle and material fabrication⁴¹'⁴³.

[0177] AimlC. Evaluate the therapeutic efficacy with human keratinocytes. In this aim, the inventors will use the 3-D human skin organotypic culture model to evaluate the therapeutic efficacy of RNA replicon vectors for STS delivery. The inventors have generated lentiviral vector encoding human STS gRNA for CRISPR-based knockout in primary human keratinocytes (Fig. 4).

[0178] Human skin 3-D organoid culture: The inventors will collaborate with STEM (skin tissue engineering & morphology) core to develop human skin 3-D organoid culture with WT and STS KO cells. The raft culture will be prepared by seeding the cells to collagen hydrogel and then lift to air-liquid interface for maturation via established protocol^65,66. Successful stratification of the organoid culture will be verified by tissue histology. RNA and LNP mixture will be delivered to mature skin organoids by injection as described in Aim IB. Samples will be collected at different time points up to 4 weeks after injection. Expression of STS will be evaluated by IHC, and potential changes in skin architecture will be determined by histology analysis. All samples will be analyzed and scored in blinded studies through the UChicago Pathology Core.

[0179] Potential outcomes and caveats: The inventors expect to see durable expression of STS in human keratinocytes with engineered RNA replicon vectors. Although unlikely, if STS deletion affects skin stratification in the organoid model, the inventors will use inducible Cas9 vector for STS KO in human keratinocytes ⁶⁷.

[0180] Specific Aim2: Explore the molecular mechanism for enhanced expression with the RNA replicon vector.

[0181] Although different RNA replicons have been developed for gene delivery, it remains largely unclear how RNA replicon leads to cytopathic effects and it expression is controlled in host cells. Understanding the underlying molecular mechanism will provide essential guidance to optimize the vector design for STS_Sindbis virus or replicon can inhibit host cell protein synthesis, which can lead to cytotoxicity^22,55,56. P726S mutation can reduce this inhibition. To monitor this process, the inventors will label mouse keratinocytes transfected with different luciferase encoding RNAs (mRNA, WT replicon, P726S replicon, N252S/T564A replicon, or mixture of P726S and N252S/T564A at 1 :3 ratio). Cells will be incubated in medium without methionine for 30 minutes, and then subjected to a pulse label with ³⁵ S-m ethionine. De novo protein synthesis will be determined by SDS-PAGE. [0182] Innate immune responses. Exposure of cells to RNA replicon can induce innate immune responses such as interferon (IFN) expression^21,23. Suppression of IFN signaling can prolong cargo gene expression⁶⁸, the inventors will examine expression of both IFNa and IFNfl in transfected mouse keratinocytes by ELISA and RT- analysis.

[0183] RNA-seq based analysis of host responses, the inventors will collect total RNA samples from mouse keratinocytes. Upon RNA extraction by RNeasy kit (Qiagen), DNA and rRNA from total RNA will be removed using DNAse-I and Ribo-zero (Illumina) and then samples with an RNA integrity value of >7 will be used for RNA-seq. The inventors will use Illumina Scriptseq for library prep and paired end 150 bp SBS Chemistry to generate sequences using our genomics core Illumina HiSeq4000 platform. Sixty million reads will be generated for each sample. Quality filtering of reads will be done using FastQC (vO.11.2) and then sequences will be mapped to the reference genome using TopHat2 (v2.1.0). The transcript assembly and calculation of differential gene expression. The Cuffmerge (vl.0.0) tool will be used to merge and generate a combined transcript. The merged transcript and reference genome fasta will be used by the Cuffdiff (v2.2.1) tool to generate differential gene expression⁶⁹. Gene function and pathway analysis will be performed by the Ingenuity Pathway Analysis tool (Qiagen Inc.). Cutoff parameters used for IPA analysis will be from either sample’s FPKM value as >=1, Log2 Fold expression difference >= 2, p-value < 0.05. There will be a false discovery rate of q < 0.01, to identify differentially expressed genes⁷⁰. The inventors will us R- packages Limma for vennDiagram & heatmap.2 from ggplots/gplots for plotting heatmaps. The inventors regulated genes, and these results will be analyzed for biological pathway analysis, to determine the impact of RNA replicons.

Example 2

[0184] Replicons are self-amplifying recombinant RNA molecules expressing proteins sufficient for their own replication, but which do not produce infectious particles. Replicons can persist in cells and are passed on during cell division, enabling quick, efficient and high throughput testing of drug candidates that act on viral transcription, translation and replication. Safety of viral replicons has made them a popular tool for antiviral R&D and have been used to investigate a broad spectrum of antiviral compounds. To engineer RNA vectors for durable expression in skin, we have designed a continuous directed evolution platform with skin keratinocytes by leveraging the intrinsic mutagenic potential of RNA replicon vectors. With this innovative platform, we have identified a novel Venezuelan equine encephalitis (VEE) replicon, which can greatly enhance expression of “cargo” genes under the subgenomic promoter in vitro and in vivo. However, transduction of VEE RNA replicion into host cells can induce strong innate immune response, which can abolish or significantly reduce expression VEE RNA. In this patent, we tested the novel approach by engineering novel VEE replicon, which encodes both a cargo gene of interest together with a viral protein to suppress cellular immune response. The viral genes include:

1. HCV NS3 and NS4

2. Kaposi sarcoma associated herpesvirus ORF52

3. HCV NS5A

4. zika virus NS2A

[0185] Our data show that co-expression with HCV NS3/4 fusion protein can significantly elongate expression of replicon RNA in vivo.

[0186] Materials And Methods

[0187] RNA preparation. To generate replicon RNA, the engineered replicon plasmids were linearized using the restriction enzyme Mlu I and RNA synthesized using Hi Scribe® T7 ARCA mRNA Kit (with tailing) (NEB, #E2060S) and RNA was purified with the Monarch® RNA Cleanup Kit (NEB, # T2050S) according to the manufacturer’s protocol. With in vitro transcription, we prepared 50 g each of control replicon RNA and stored under -80°C freezer. [0188] Lipid nanoparticle preparation. We prepared MC3 LNP, which is composed of ionizable lipid DLin-MC3-DMA, helper lipid (1,2-DSPC), cholesterol and DMG-PEG2000 with a molar ratio of 50: 10:38.5: 1.5. The lipid mixture is dissolved in ethanol and RNA is dissolved in 50mM sodium acetate buffer (pH 5.0).

[0189] RNA injection. We injected replicon RNA or vehicle (LNP) intradermally (3 ug RNA per mouse) on the lower limbs and intravenously (10 ug RNA per mouse) via tails into nude mice or BALB/c mice (3 animals for each experimental group, 3 for control group, 8-10 weeks old, both sexes). Each animal received one dose and were monitored through Bioluminescence Imaging (BLI) system each week for one month.

[0190] In vivo imaging. Each group was anesthetized, following appropriate IACUC guidelines in isoflurane anesthesia, and imaged 5 min after intraperitoneal administration of 125 nmol FFZ reconstituted in 50 pL of room temperature DPBS. Images were acquired using an I VIS spectrum with imaging parameters set to: FOV D, 20 cm, binning = 16, exposure = 60 sec, and f/stop = 1.2. The stage was heated to 37°C and a series of images acquired at 10 and 15 min post-i.p. injection of the substrates to determine the timing for maximal signal output. Bioluminescent signals were then quantified by spectral unmixing. The signal intensity was represented by the radiance unit of photons (p) /sec/cm²/sr.

[0191] Results [0192] Generation of NanoLuc luciferase (NLuc) expressing reporter VEE replicon

[0193] To generate autonomously replicating recombinant VEE replicon expressing a reporter gene, we cloned VEE nsPl-nsP4 genes (nucleotide 1-7526) behind a strong T7 promoter for generating high yields of transcript, and includes a 5’ UTR, 3’ UTR, and a poly A tail. The NLuc (nano luciferase) open reading frame (ORF) was inserted downstream the VEE nsP genes, and its expression will be driven by the subgenomic promoter of VEE replicon. To suppress cellular immune response to viral RNA, we inserted various different viral genes behind the NLuc coding sequence and a T2A self cleavage peptide for co-expression (Fig. 5).

[0194] Long-term tracking of live imaging of NLuc expressing after intradermal and intravenous injection

[0195] To evaluate the dynamics of NLuc expression in mice, we intradermally injected adult mice in the lower limbs with either mRNA of NLuc (control), VEE-NLuc or VEE-NLuc- with different viral anti-immune genes encapsulated by MC3 LNP. Intravital bioluminescence imaging show that co-expression with HCV NS3/4 can significantly elongate NLuc expression in vivo (Fig. 6). Co-expression with Kaposi sarcoma herpesvirus ORF52, Zika NS2A, or HCV NS5A can also elongate NLuc expression in vivo, compared with NLuc mRNA (Fig. 7). To test potential delivery of replicon RNA to other organs, we performed intravenous injection of control or different VEE replicon RNA to immunocompetent CD1 mice. VEE replicon with HCV NS3/4 demonstrated significantly more durable expression of luciferase in liver after injection (Fig. 8).

[0196] Expression of different cargo genes with the replicon vectors

[0197] To determine the expression of different GOI (gene of interest) with the replicon vector, we tested expression of different genes, including Filaggrin, Col7Al, STS, IL23 decoy receptor, and IL 10 in cultured keratinocytes in vitro. VEE replicon vector with HCV NS3/4 exhibited most durable expression of different cargo genes in vitro (Fig. 9). Filaggrin and STS are mutated in human ichthyosis, Col7Al is mutated in human with RDEB (recessive dystrophic epidermolysis bullosa). IL 10 and IL23 decoy receptor can potentially suppress tissue inflammation, and can be used for treatment of skin inflammatory diseases, such as psoriasis. Together, our data suggest that the evolved replicon vectors can have broad applications for different diseases, including genetic disorders and autoimmune diseases.

Example 3

[0198] A dual-vector, self-amplifying RNA replicon system based on the Sindbis Virus (SINV) can restore functional COL7A1 and promote wound healing in recessive dystrophic epidermolysis bullosa (RDEB) patients. [0199] Disease Overview

[0200] Etiology: Recessive dystrophic epidermolysis bullosa (RDEB) is the most severe form of dystrophic EB caused by autosomal recessive inheritance of mutated COL7A1 which encodes pro-al(VII), a component required for the assembly of type VII collagen. Loss of function of COL7A1 depletes type VII collagen, inhibiting formation of structural anchoring fibrils in the skin’s epidermal basement membrane zone, causing skin layer separation in response to minor trauma, and ultimately severe chronic blistering

[0201] Epidemiology: RDEB incidence estimated at 0.1-6.65 per million births, with estimated prevalence of 3.5-20.4 per million people worldwide. -3000 patients currently living with RDEB in the US

[0202] Current treatments: Krystal Biotech’s Vyiuvek (approved 2023) is an HSV-1 based COL7A1 gene replacement therapy applied topically Q1W. Abeona Therapeutic’s EB- 101 (P3) is an autologous engineered ex vivo cell therapy that integrates functional COL7A1 into epidermal sheets that are grafted onto wounds

[0203] Unmet need: RDEB patients suffer chronic skin blistering and non-healing wounds. Current Tx promote wound healing but cannot prevent new wounds and require frequent administration or a complex skin graft

[0204] The Novelty and Innovation

[0205] Ambition: Cost-effective, durable gene delivery system to restore COL7A1 expression in RDEB patients to (1) prevent new lesions in intact skin (Vyjuvek is only for open wounds) & (2) Q1M use (Vyjuvek use is Q1W)

[0206] Novelty: Dual SINV-based self-amplifying replicon vectors packaged in the lipid nanoparticle MC3 delivered either intradermally or topically to restore type VII collagen production in RDEB

[0207] Features: Directed evolution produced two SINV replicon vectors, each with mutations which are effective in trans. The first replicon’s mutation, P726S, reduces cytopathic effects of SINV-based vectors while the combination of this and the second replicon’s N252S/T564A mutations improves durable target expression. Either replicon alone exhibits diminishes in vivo and in vitro expression. A 3: 1 ratio can result in enhanced, durable longterm cargo gene expression

[0208] Indication expansion: This topical gene delivery system could be applied to other genetic skin diseases or possibly in other tissues such as the lungs [0209] Mutant SINV replicons: Keratinocytes were transfected with SINV replicon and exposed to replication stress pressure. Two mutant replicons acting in trans were derived from surviving cells

[0210] Luciferase expression: Durable expression was observed in nude mice up to 5 weeks after intradermal injection

[0211] Topical administration: Topical formulation showed durable luciferase expression in nude mice up to 5 weeks post-delivery

* * *

[0212] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

WHAT IS CLAIMED IS:

1. An RNA replicon comprising one mutation in nonstructural protein 3 (nPs3) of the RNA replicon and one mutation in nonstructural protein 4 (nPs4) of the RNA replicon.

2. The RNA replicon of claim 1, wherein the mutation in nPs3 comprises an N252S mutation.

3. The RNA replicon of claim 1, wherein the mutation in nPs4 comprises a T564A mutation.

4. The RNA replicon of claim 1, wherein the RNA replicon is a Sindbis RNA replicon.

5. The RNA replicon of claim 1, wherein the RNA replicon encodes a protein of interest.

6. The RNA replicon of claim 5, wherein the protein of interest is a protein deficient in a genetic disease, a cancer, an autoimmune disease, an infection, and/or any other disease caused by or amplified by a deficient protein.

7. The RNA replicon of claim 5, wherein the protein of interest is mutated and/or deleted in a genetic disease, a cancer, an autoimmune disease, an infection, and/or any other disease caused by or amplified by a deficient protein.

8. The RNA replicon of claim 5, wherein the protein of interest is a steroid sulfatase (STS).

9. The RNA replicon of claim 6, wherein the genetic disease is a recessive X-linked ichthyosis.

10. The RNA replicon of claim 6, further comprising at least one viral protein, wherein the viral protein is capable of suppressing a cellular immune response.

11. The RNA replicon of claim 10, wherein the viral protein is hepatitis C nonstructural protein 3, hepatitis C nonstructural protein 4, hepatitis C nonstructural protein 5A, Ebola virus VP35, Measles V antigen, Kaposi sarcoma associated herpesvirus ORF52, influenza H7N9 nonstructural protein 1, vaccinia Bl 8R, Dengue virus 3 nonstructural protein 5, herpes simplex virus- 1 infected cell protein 1, zika virus nonstructural protein 2 A, or a combination thereof.

12. A self-replicating RNA molecule encoding nonstructural proteins comprising one mutation in nonstructural protein 3 (nPs3) of the self-replicating RNA molecule and one mutation in nonstructural protein 4 (nPs4) of the self-replicating RNA moelcule.

13. The self-replicating RNA molecule of claim 12, wherein the mutation in nPs3 comprises an N252S mutation.

14. The self-replicating RNA molecule of claim 12, wherein the mutation in nPs4 comprises a T564A mutation.

15. The self-replicating RNA molecule of claim 12, wherein the self-replicating RNA molecule is a Sindbis RNA replicon.

16. The self-replicating RNA molecule of claim 12, wherein the self-replicating RNA molecule encodes a protein of interest.

17. The self-replicating RNA molecule of claim 12, further comprising at least one viral protein, wherein the viral protein is capable of suppressing a cellular immune response.

18. The self-replicating RNA molecule of claim 17, wherein the viral protein is hepatitis C nonstructural protein 3, hepatitis C nonstructural protein 4, hepatitis C nonstructural protein 5A, Ebola virus VP35, Measles V antigen, Kaposi sarcoma associated herpesvirus ORF52, influenza H7N9 nonstructural protein 1, vaccinia Bl 8R, Dengue virus 3 nonstructural protein 5, herpes simplex virus- 1 infected cell protein 1, zika virus nonstructural protein 2A, or a combination thereof.

19. The self-replicating RNA molecule of claim 16, wherein the protein of interest is a protein deficient in a genetic disease, a cancer, an autoimmune disease, an infection, and/or any other disease caused by or amplified by a deficient protein.

20. The self-replicating RNA molecule of claim 16, wherein the protein of interest is mutated and/or deleted in a genetic disease, a cancer, an autoimmune disease, an infection, and/or any other disease caused by or amplified by a deficient protein.

21. The self-replicating RNA molecule of claim 17, wherein the protein of interest is a steroid sulfatase (STS).

22. The self-replicating RNA molecule of claim 19, wherein the genetic disease is a recessive X-linked ichthyosis.

23. An RNA replicon encoding at least one viral protein, wherein the viral protein is capable of suppressing a cellular immune response.

24. The RNA replicon of claim 23, wherein the viral protein is hepatitis C nonstructural protein 3, hepatitis C nonstructural protein 4, hepatitis C nonstructural protein 5A, Ebola virus VP35, Measles V antigen, Kaposi sarcoma associated herpesvirus ORF52, influenza H7N9 nonstructural protein 1, vaccinia Bl 8R, Dengue virus 3 nonstructural protein 5, herpes simplex virus- 1 infected cell protein 1, zika virus nonstructural protein 2 A, or a combination thereof.

25. The RNA replicon of claim 23, wherein the replicon is a Venezuelan equine encephalitis replicon.

26. The RNA replicon of claim 23, wherein the replicon comprises the sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

27. The RNA replicon of claim 23, wherein the replicon comprises a nucleic acid encoding a protein of interest.

28. The RNA replicon of claim 27, wherein the protein of interest is COL7A1, a steroid sulfatase, IL- 10, an IL-23 decoy receptor, or filaggrin.

29. An RNA replicon comprising the nucleic acid of SEQ ID NO: 1.

30. An RNA replicon comprising the nucleic acid of SEQ ID NO:2.

31. An RNA replicon comprising the nucleic acid of SEQ ID NO:3.

32. An RNA replicon comprising the nucleic acid of SEQ ID NO:4.

33. An RNA replicon comprising the nucleic acid of SEQ ID NO:5.

34. An RNA replicon comprising the nucleic acid of SEQ ID NO:6.

35. An expression construct comprising the RNA replicon of any one of claims 1 to 11 and/or 23 to 34 and/or the self-replicating RNA molecule of any one of claims 12 to 22.

36. A vaccine comprising the RNA replicon of any one of claims 1 to 11 and/or 23 to 34, the self-replicating RNA molecule of any one of claims 12 to 22, and/or the expression construct of claim 36.

37. A gene delivery platform comprising the RNA replicon of any one of claims 1 to 11 and/or 23 to 34, the self-replicating RNA molecule of any one of claims 12 to 22, and/or the expression construct of claim 35.

38. A therapeutic composition comprising the RNA replicon of any one of claims 1 to 11 and/or 23 to 34, the self-replicating RNA molecule of any one of claims 12 to 22, and/or the expression construct of claim 35.

39. The therapeutic composition of claim 38, further comprising a nanoparticle.

40. The therapeutic composition of claim 39, wherein the nanoparticle comprises an ionizable lipid, a helper lipid, a sterol, and a DMG-PEG.

41. The therapeutic composition of claim 40, wherein the ionizable lipid comprises D-Lin- MC3-DMA, wherein the helper lipid comprises (1,2-DSPC), wherein the sterol comprises cholesterol, and/or wherein the DMG-PEG comprises DMG-PEG2000.

42. The therapeutic composition of any one of claims 38 to 41, wherein the therapeutic composition is formulated for topical administration or intravenous administration.

43. A method of treating a disease in a patient, the method comprising administering to the patient an effective amount of the RNA replicon of any one of claims 1 to 11 or 23 to 34, the self-replicating RNA molecule of any one of claims 12 to 22, the expression construct of claim 35, the vaccine of claim 36, the gene delivery platform of claim 37, and/or the therapeutic composition of any one of claims 38 to 41.

44. The method of claim 43, wherein the patient has, is suspected of having, or is diagnosed with having a deficiency and/or mutation in a specific protein.

45. The method of claim 44, wherein the specific protein is a steroid sulfatase.

46. The method of claim 43, wherein the RNA replicon encodes a protein of interest, and wherein the patient has, is suspected of having, or is diagnosed with having a deficiency and/or mutation in the protein of interest and/or the patient has, is suspected of having, or is diagnosed with having a disease associated with the protein of interest.

47. The method of claim 46, wherein the protein of interest is a steroid sulfatase.

48. The method of any one of claims 43 to 47, wherein the patient has, is suspected of having, or has been diagnosed with having a genetic disease, a cancer, an autoimmune disease, an infection, and/or any other disease caused by or amplified by a deficient protein.

49. The method of claim 48, wherein the genetic disease, the cancer, the autoimmune disease, the infection is caused by or amplified by a deficient protein.

50. The method of claim 49, wherein the deficient protein comprises a mutated protein and/or deleted protein.

51. The method of any one of claims 43 to 50, wherein the patient has, is suspected of having, or has been diagnosed with having a recessive X-linked ichthyosis.

52. The method of any one of claims 43 to 50, wherein the patient has, is suspected of having, or has been diagnosed with having dystrophic epidermolysis bullosa.

53. A method of treating X-linked ichthyosis in a patient, the method comprising administering an RNA replicon encoding a steroid sulfatase and a viral protein to the patient.

54. A method of treating dystrophic epidermolysis bullosa in a patient, the method comprising administering an RNA replicon encoding Col7Al and a viral protein to the patient.

55. A method of increasing expression of a protein of interest in a cell of interest, the method comprising contacting the cell with the RNA replicon the RNA replicon of any one of claims 1 to 11 and/or 23 to 33, the self-replicating RNA molecule of any one of claims 12 to 22, the expression construct of claim 35, the vaccine of claim 36, the gene delivery platform of claim 37, and/or the therapeutic composition of any one of claims 38 to 41.

56. The method of claim 55, wherein the cell of interest is a skin cell.

57. The method of claim 55, wherein the cell of interest is a liver cell.