WO2025085494A1

WO2025085494A1 - Treatment of fibronectin-related inflammation, fibrosis and neuropathic pain conditions

Info

Publication number: WO2025085494A1
Application number: PCT/US2024/051530
Authority: WO
Inventors: Yu-Shang LEE; Ching-Yi Lin
Original assignee: Cleveland Clinic Foundation
Current assignee: Cleveland Clinic Foundation
Priority date: 2023-10-17
Filing date: 2024-10-16
Publication date: 2025-04-24
Anticipated expiration: 2026-04-17

Abstract

Provided herein are compositions, systems, kits, and methods for treating fibronectin-related inflammation and/or fibrosis which, for example, occurs in pain (e.g., neuropathic pain, inflammatory pain, chemotherapy-induced peripheral neuropathy, etc.), nervous system disorders (e.g., stoke, brain injury, spinal cord injury, peripheral nerves injury, etc.), neurodegenerative diseases (e.g., Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, etc.), organ transplantation (e.g., lung, kidney, heart, liver, etc.), lung fibrosis, liver fibrosis, amputation, and cosmetic and plastic surgery applications, in a subject by administering (e.g., subcutaneously) a fibronectin reduction peptide (FRP), or a nucleic acid sequence encoding the FRP, wherein the FRP comprises: i) a cell membrane and blood brain barrier (BBB) penetrating (CMBBBP) motif, ii) a fibronectin binding motif, and iii) a lysosome targeting motif.

Description

TREATMENT OF FIBRONECTIN-RELATED INFLAMMATION, FIBROSIS AND NEUROPATHIC PAIN CONDITIONS

The present application claims priority to U.S. provisional application serial number 63/590,804, filed October 17, 2023, which is herein incorporated by reference in its entirety.

SEQUENCE LISTING

The text of the computer readable sequence listing filed herewith, titled “CCF_42468_601_SequenceListing.xml”, created October 16, 2024, having a file size of 58,655 bytes, is hereby incorporated by reference in its entirety.

FIELD

Provided herein are compositions, systems, kits, and methods for treating fibronectin- related inflammation and/or fibrosis which, for example, occurs in pain (e.g., neuropathic pain, inflammatory pain, chemotherapy-induced peripheral neuropathy, etc.), nervous system disorders (e.g., stoke, brain injury, spinal cord injury, peripheral nerves injury, etc.), neurodegenerative diseases (e.g., Alzheimer’s disease, amyotrophic lateral sclerosis, multiple sclerosis, etc.), organ transplantation (e.g., lung, kidney, heart, liver, etc.), lung fibrosis, liver fibrosis, amputation, and cosmetic and plastic surgery applications, in a subject by administering (e.g., subcutaneously) a fibronectin reduction peptide (FRP), or a nucleic acid sequence encoding the FRP, wherein the FRP comprises: i) a cell membrane and blood brain barrier (BBB) penetrating (CMBBBP) motif, ii) a fibronectin-binding motif, and iii) a lysosome-targeting motif.

BACKGROUND

Over 100 million Americans suffer from chronic pain, which causes staggering economic costs in the United States (U.S.), especially the burdens of healthcare and lost productivity. The classic symptoms of chronic pain are allodynia and hyperalgesia¹. Allodynia is a condition in which pain is caused by a stimulus that does not normally elicit pain, such as a bad sunburn which can cause temporary allodynia, and touching sunburned skin, or running cold or warm water over it, can be very painful. Hyperalgesia is an abnormally increased sensitivity to pain, which may be caused by damage to nociceptors or peripheral nerves and can cause hypersensitivity to stimulus. The Centers for Disease Control estimates that 20.4% (50 million) of U.S. adults endure chronic pain, and 8.0% (19.6 million) have high-impact chronic pain (i.e. pain lasting longer than six months with substantial activity restrictions)². A lack of diverse and effective therapeutic options has led physicians to rely on opiates to treat patients with chronic pain³. Opioids are thus the most widely prescribed chronic pain medications. However, while they are excellent for managing moderate to severe pain, opioids are beset with obvious side effects⁴. In 2016, an estimated 42,249 Americans died from an opioid overdose, and 1 in 4 patients receiving opioid prescriptions is now dependent^3,5. Furthermore, in 2017, 1.7 million people in the U.S. suffered from opioid use disorder (OUD) due to prescription of opioid pain medication⁶. Therefore, alternative non-opioid analgesics are urgently needed to curtail OUD for chronic pain treatment.

SUMMARY

Provided herein are compositions, systems, kits, and methods for treating fibronectin- related inflammation and/or fibrosis which, for example, occurs in pain (e.g., neuropathic pain, inflammatory pain, chemotherapy-induced peripheral neuropathy, etc.), nervous system disorders (e.g., stoke, brain injury, spinal cord injury, peripheral nerves injury, etc.), neurodegenerative diseases (e.g., Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, etc.), organ transplantation (e.g., lung, kidney, heart, liver, etc.), lung fibrosis, liver fibrosis, amputation, and cosmetic and plastic surgery applications, in a subject by administering (e.g., subcutaneously) a fibronectin reduction peptide (FRP), or a nucleic acid sequence encoding the FRP, wherein the FRP comprises: i) a cell membrane and blood brain barrier (BBB) penetrating (CMBBBP) motif, ii) a fibronectin binding motif, and iii) a lysosome targeting motif.

In some embodiments, provided herein are methods of treating fibronectin-related inflammation and/or fibrosis which occurs, for example, in pain (e.g., neuropathic pain, inflammatory pain, chemotherapy-induced peripheral neuropathy, etc.), nervous system disorders (e.g., stoke, brain injury, spinal cord injury, peripheral nerves injury, etc.), neurodegenerative diseases (e.g., Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, etc.), organ transplantation (e.g., lung, kidney, heart, liver, etc.), lung fibrosis, liver fibrosis, amputation, and cosmetic and plastic surgery applications, in a subject comprising: administering a fibronectin reduction peptide (FRP), or a nucleic acid sequence encoding the FRP (e.g., in a vector or as mRNA, such as in lipid nanoparticles), to a subject with fibronectin-related inflammation and/or fibrosis which occurs in pain (e.g., neuropathic pain, inflammatory pain, chemotherapy-induced peripheral neuropathy, etc.), nervous system disorders (e.g., stoke, brain injury, spinal cord injury, peripheral nerves injury, etc.), neurodegenerati e diseases (e.g., Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, etc.), organ transplantation (e.g., lung, kidney, heart, liver, etc.), lung fibrosis, liver fibrosis, amputation, and cosmetic and plastic surgery application, wherein the FRP comprises: i) a cell membrane and blood brain barrier penetrating (CMBBBP) motif, ii) a fibronectin binding motif, and iii) a lysosome targeting motif.

In particular embodiments, provided herein are compositions, systems, and kits comprising: a fibronectin reduction peptide (FRP) or a nucleic acid sequence encoding the FRP (e.g., in a vector or as mRNA, such as in lipid nanoparticles), as described above and herein. Tn particular embodiments, the systems and kits further comprise a device for injecting a composition comprising the FRP into a subject's peripheral nervous system, optionally, wherein the injecting is subcutaneous.

In additional embodiments, the subject has fibronectin-related neuropathic pain. In particular embodiments, the neuropathic pain is localized to at least one peripheral nerve of the subject. In particular embodiments, the administering reduces the level of fibronectin present at the at least one peripheral nerve. In some embodiments, the administering is performed subcutaneously, optionally in the area of a peripheral nerve of the subject. In other embodiments, the administering is performed intravenously, intraperitoneally, intramuscularly, intrathecally, orally, or intraocularly.

In certain embodiments, the administering is repeated daily for at least 3 days (e.g., at least 3, 4, 5, 6, 7, 8, or 9), optionally near an injured nerve, and optionally for at least 10 days (e.g., at least 10, 11, 12, 13, or 14 days, or longer). In other embodiments, the administering is conducted after at least two days of initiation of the fibronectin-related inflammatory and/or neuropathic pain condition. In some embodiments, the administering is conducted after at least one weeks (e.g., at least 2, 3, 4, or 5 weeks, or longer) of initiation of the fibronectin-related inflammation, fibrosis and/or neuropathic pain condition.

In particular embodiments, the CMBBBP motif is located at the N-terminal or C- terminal of the FRP, or optionally at neither the N or C terminals. In other embodiments, the lysosome targeting motif is located at the N-terminal or C-terminal of the FRP, or optionally at neither the N or C terminals. In particular embodiments, the fibronectin binding motif is located between another two motifs, or optionally located at the N-terminal or C-terminal of the FRP.

In some embodiments, the CMBBBP motif comprises or consists of an amino acid sequence shown in SEQ ID NOS:1 or 5-27, or a sequence with one, two or more amino acid additions, subtractions (e.g., from N and/or C terminal), or substitutions. In other embodiments, the fibronectin binding motif comprises or consists of an amino acid sequence shown in SEQ ID NOS:2 or 28-47, or a sequence with one, two or more amino acid additions, subtractions (e.g., at the N and/or C terminals), or substitutions. In particular embodiments, the lysosome targeting motif comprises or consists of an amino acid sequence shown in SEQ ID NO:3 or 48-56, or a sequence with one, two or more amino acid additions, subtractions (e.g., at N and/or C terminals), or substitutions. In other embodiments, the FRP comprises or consists of an amino acid sequence shown in SEQ ID NO:4 or 57-67, or a sequence with one, two or more amino acid additions, subtractions (e.g., at N or C terminals), or substitutions.

DESCRIPTION OF THE FIGURES

Figure 1 shows a schematic drawing how an exemplary FRP reduces injury- upregulated fibronectin by directing specifically-bound fibronectin to a lysosome for degradation in a variety of clinical indications associated with fibronectin-related inflammation and fibrosis.

Figure 2 shows a schematic representation of experimental procedures to test the efficacy of the one-week delayed treatment of an exemplary FRP on decreasing chronic neuropathic pain associated behaviors including thermal hyperalgesia, mechanical allodynia and mechanical hyperalgesia, in chronic constriction injury (CCI) rats.

Figure 3 shows a biodistribution of FITC-labeled FRP (FITC-FRP) within the injured sciatic nerve of rats. Subcutaneous injection of FITC-FRP was administered beginning 7 days after CCI for an additional 7 days. Injured sciatic nerve was harvested for immunohistochemistry. Representative images demonstrating FITC-labeled FRP (green) was distributed surrounding the injury epicenter of sciatic nerve.

Figure 4 shows fibronectin levels were significantly upregulated in the sciatic nerve near the injured site after CCI of sciatic nerve. Representative images of fibronectin immunostaining (red) from uninjured sciatic nerve (A) and CCI injured sciatic nerves (B, C, D). Injured sciatic nerves showed significantly-increased fibronectin compared to uninjured sciatic nerve (A), at 1 week post-CCI (B) and continuously increased at 3 weeks post-CCI (C). Such increases in fibronectin were significantly reduced with one-week delayed FRP treatment (D, E). Graphs (E) represent mean ± SEM (N=6 per group) for the fibronectin intensity. **, P < 0.01; ***, P < 0.001 when compared to the non-injury control; +, P < 0.05 when compared to the scrambled peptide treated CCI group.

Figure 5 shows fibronectin levels were significantly upregulated in the dorsal root ganglion (DRG) after CCI of sciatic nerve. Representative images of fibronectin immunostaining (red) in the DRG collected from non-injury control (A) and CCI groups (B, C, and D). Fibronectin in DRG was significantly upregulated in the CCI animals at 1 week post-CCI (B) and continuously increased at 3 weeks post-CCI (C). Such increases in fibronectin were significantly reduced with one-week delayed FRP treatment (D, E). Graphs (E) represent mean + SEM (N=6 per group) for the fibronectin intensity. *, P < 0.05; **, P < 0.01 when compared to the non-injury control; ++, P < 0.01 when compared to the scrambled peptide treated CCI group.

Figure 6 shows treatment with an exemplary FRP attenuates thermal hyperalgesia in CCI rats. Animals were treated daily with FRP for 2 weeks, beginning 1 week after CCI. An automated Hargreaves Apparatus was used to provide noxious heat and collect data. FRP- treated animals (CCI+FRP) showed significant decreases in hindpaw thermal hyperalgesia, as indicated by increased maximum tolerated latency (sec.) needed for hindlimb withdrawal, compared to scrambled peptide-treated animals (CCI+scrambled peptide) over the entire observation period. Values represented mean + SEM, n=12 per group.

Figure 7 shows treatment with the exemplary FRP attenuates mechanical allodynia in CCI rats. Animals were treated daily with the FRP for 2 weeks, beginning 1 week after CCI. An automated dynamic plantar aesthesiometer was used to provide innocuous touch and collect data⁸. FRP-treated animals (CCI+FRP) showed significant decreases in hindpaw mechanical allodynia, as indicated by increased maximum tolerated force (g, A) and latency (sec., B) needed for hindlimb withdrawal, compared to scrambled peptide-treated animals (CCI+scrambled peptide) over the entire observation period. Graphs represent the quantified analyses of cumulative addition of force and time for each group (C). **p < 0.01, ***p < 0.001, ****p < 0.0001 when compared to the scrambled peptide treated CCI group, two-way ANOVA. Values represented mean + SEM, n=12 per group.

Figure 8 shows treatment with the exemplary FRP attenuates mechanical hyperalgesia in CCI rats. Animals were treated daily with FRP for 2 weeks, beginning 1 week after CCI. An automated dynamic plantar aesthesiometer was used to provide noxious touch and collect data⁸. FRP-treated animals (CCI+FRP) showed significant decreases in hindpaw mechanical hyperalgesia, as indicated by increased maximum tolerated force (g, A) and latency (sec., B) needed for hindlimb withdrawal, compared to scrambled peptide-treated animals (CCI+scrambled peptide) over the entire observation period. Graphs represent the quantified analyses of cumulative addition of force and time for each group (C). **p < 0.01 , ***p < 0.001, ****p < 0.0001 when compared to the scrambled peptide treated CCI group, two-way ANOVA. Values represented mean + SEM, n=12 per group.

DETAILED DESCRIPTION Provided herein are compositions, systems, kits, and methods for treating fibronectin- related inflammation and/or fibrosis which, for example, occurs in pain (e.g., neuropathic pain, inflammatory pain, chemotherapy-induced peripheral neuropathy, etc.), nervous system disorders (e.g., stoke, brain injury, spinal cord injury, peripheral nerves injury, etc.), neurodegenerative diseases (e.g., Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, etc.), organ transplantation (e.g., lung, kidney, heart, liver, etc.), lung fibrosis, liver fibrosis, amputation, and cosmetic and plastic surgery applications, in a subject by administering (e.g., subcutaneously) a fibronectin reduction peptide (FRP), or a nucleic acid sequence encoding the FRP, wherein the FRP comprises: i) a cell membrane and blood brain barrier (BBB) penetrating (CMBBBP) motif, ii) a fibronectin binding motif, and iii) a lysosome targeting motif.

Integrins, the receptors for extracellular matrix (ECM) including fibronectin, are found upregulated on sensory neurons that mediate pain in a variety of inflammatory and neuropathic pain conditions. The therapeutic potential of inhibiting fibronectin/integrin signaling for pain treatment has been demonstrated by the reports that (1) treatment of a4pi integrin function-blocking antibody decreases mechanical allodynia elicited by spinal cord injury (SCI); (2) acute intra-spinal cord injection of fibronectin or its alternatively spliced isoform, connecting segment 1 (CS1), prevents the development of chronic allodynia after SCI; (3) acute intra-sciatic nerve injection of fibronectin CS1 attenuates mechanical allodynia after injury in rat sciatic nerve. These studies suggest that decreasing signaling mediated by fibronectin/integrin, the critical targets within inflammation, fibrosis and/or pain pathways, is an effective approach to manage neuropathic pain after neurotrauma. There are, however, no clinically available approaches to effectively decrease fibronectin/integrin signaling.

To pursue an opioid-alternative and promising therapeutic approach to treat chronic neuropathic pain, we have developed an innovative small peptide, called fibronectin reduction peptide (FRP). FRP (SEQ ID NO:4) is 35 amino acids in length and consists of three unique motifs, including an N-terminal cell membrane and blood brain barrier penetrating motif (SEQ ID NO:1), a central fibronectin binding motif (SEQ ID NO:2), and a C-terminal lysosome targeting motif (SEQ ID NO:3). These specific elements conceptually allow FRP to: (1) efficiently distribute into the nervous system via its N-terminal motif; (2) specifically bind to fibronectin via its fibronectin binding motif; and (3) degrade fibronectin- FRP complexes in the lysosome via its C-terminal motif (Figure 1). These elements are critical and novel because FRP is a translatable and specific approach to target fibronectin/integrin pathways that are important for inflammatory, fibrotic and/or nociceptive responses. In certain embodiments, the FRP has the following sequence:

N - YGRKKRRQRRR-GAPGSSYWTG-KFERQKILDQRFFE- C (SEQ ID NO:4). One of skill in the art could construct a corresponding nucleic acid sequence based on the known codon triplets for the amino acids specified in SEQ ID NO:4. The SEQ ID NO:4 sequence has the following three components:

A cell membrane and blood brain barrier (BBB) penetrating (CMBBBP): YGRKKRRQRRR (SEQ ID NO:1);

A fibronectin binding motif: GAPGSSYWTG (SEQ ID NO:2); and

A lysosome targeting motif: KFERQKILDQRFFE (SEQ ID NO:3).

Each of these motifs may be constructed with longer, shorter, or mutated versions of the sequences shown in SEQ ID NOS: 1-3 and 5-56. For example, one could change one, two, three (or more) amino acids in these sequences. For example, one could make conservative changes to a particular amino acid sequence. Conservative amino acid substitutions refer to the interchangeability of residues having side chains with similar chemical and physical properties. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine. Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine. In certain embodiments, provided herein are peptides that have substantial identity (e.g., 95% . . . 99%) to at least a portion of the amino acid sequences shown in SEQ ID NOs:l-67

In certain embodiments, the cell-membrane, blood brain barrier, penetrating motif of an FRP is an amino acid sequence as shown in SEQ ID NOs: 1 and 5-27 of Table 1 or such a peptide with one, two, or three (or more) N-terminal or C-terminal additions, subtractions or mutations therein. One of skill in the art could construct a corresponding nucleic acid sequence based on the known codon triplets for the amino acid sequences shown in Table 1.

TABLE 1. The Blood-Brain barrier and Cell membrane penetrating motif of FRP. Amino acid (AA) sequences SEQ ID NO:

YGRKKRRQRRR 1

YGRKKRRQRRR-C 5

YGRKKRRQRRR- GC 6

CA- YGRKKRRQRRR 7

YGRKKRRQRRRYGRKKRRQRRRYGRKKRRQRRR 8

GRKKRRQRRR 9

GRKKRRQRRR-G 10

GRKKRRQRRR-CG 11

GRKKRRQRRRPP 12

GRKKRRQRRRPPQ 13

GRKKRRQRRRPPQ-C 14

RKKRRQRRR 15

RKKRRQRRR 16

RRRQRRKKR 17

RRRQRRKKR 18

GRRRRRRRRRPPQ 19

TRQARRNRRRRWRERQR-GC 20

TRRQRTRRARRNRGC 21

KLTRAQRRAAARKNKRNTR-GC 22

RRRRNRTRRNRRRVR 23

RRRRRR-GC 24 RRRRRRRR-GC 25

KETWW- ETWWTEWSQPKKKRKV 26

RQILIWFQNRRMKWKK 27

In other embodiments, the cell membrane and blood brain barrier (BBB), penetrating peptide is a protein transduction domain (PTD) with the presence of multiple arginine (R) residues as shown in SEQ ID NOs:l and 5-27 of Table 1 In other embodiments, the cell membrane, and BBB, penetrating peptide is a cell penetrating peptide (CPP) from the CPPsite 2.0, which is an updated version of database CPPsite. This site contains around 1700 unique cell penetrating peptides (CPPs) along with their secondary & tertiary structure, and can be found at www. followed by "crdd.osdd.net/raghava/cppsite/."

In certain embodiments, the fibronectin binding motif of a FRP is an amino acid sequence as shown in SEQ ID NOs:2 and 28-47 of Table 2 or such a peptide with one, two, or three (or more) N-terminal or C-terminal additions, subtractions or mutations therein. One of skill in the art could construct a corresponding nucleic acid sequence based on the known codon triplets for the amino acid sequences shown in Table 2. TABLE 2. The fibronectin binding motif of FRP.

Amino acid (AA) sequences SEQ ID NO:

GAPGSSYWTG 2

YWTG 28

SYWTG 29

SSYWTG 30

GSSYWTG 31

PGSSYWTG 32

APGSSYWTG 33

SYWTGS 34

YWTGS 35 GAPGSSYWTGS 36

GHRWKNIFYIKNENKLPTGG 37

YQDYV 38

HTTEVVGGAPQHEQIGK 39

GGPGSYFWQG 40

YFW 41

YFWQ 42

YFWQG 43

SYFWQG 44

GSYFWQG 45

PGSYFWQG 46

GPGSYFWQG 47

In certain embodiments, the lysosomal targeting motif of a FRP is an amino acid sequence as shown in SEQ ID NOs:3 and 48-56 of Table 3 or such a peptide with one, two, or three (or more) N-terminal or C-terminal additions, subtractions or mutations therein. In other embodiments, the lysosomal targeting motif is a chaperone-mediated autophagy (CMA)-targeting motif (CTM) containing a KFERQ-like motif, such as those shown in SEQ ID NOs:3 and 48-56 of Table 3. One of skill in the art could construct a corresponding nucleic acid sequence based on the known codon triplets for the amino acid sequences shown in Table 3.

TABLE 3. Lysosome targeting motif of FRP.

Amino acid (AA) sequences SEQ ID NO:

KFERQKILDQRFFE 3 KFERQ-like 48

KFERQ 49

QKILD 50

QRFFE 51

KFERQKILD 52

QKILDQRFFE 53

KFERQRFFE 54

QRFFERQ 55

QRKFERQ 56

In certain embodiments, the FRP comprises, consists essentially of, or consists of any one of the following sequences with alternative arrangement of three motifs, or a nucleic acid sequence encoding such sequences:

N - YGRKKRRQRRR-GAPGSSYWTG-KFERQKILDQRFFE - C (SEQ ID NO:4)

N - GAPGSSYWTG-YGRKKRRQRRR-KFERQKILDQRFFE - C (SEQ ID NO: 57)

N - GAPGSSYWTG-KFERQKILDQRFFE-YGRKKRRQRRR - C (SE ID NO:58)

N - YGRKKRRQRRR-KFERQKILDQRFFE-GAPGSSYWTG - C (SEQ ID NO:59)

N - KFERQKILDQRFFE-YGRKKRRQRRR-GAPGSSYWTG - C (SEQ ID NO: 60)

N - KFERQKILDQRFFE-GAPGSSYWTG-YGRKKRRQRRR - C (SEQ ID NO:61)

In other embodiments, the FRP could be shorter in length, comprises or consists of any one of the following sequences with less amino acids of three motifs, or a nucleic acid sequence encoding such sequences.

N - GRKKRRQRR- YWTGS -KFERQKILD - C (SEQ ID NO: 62)

N - YWTGS -GRKKRRQRR- KFERQKILD - C (SEQ ID NO:63)

N - YWTGS -KFERQKILD-GRKKRRQRR - C (SE ID NO:64)

N - GRKKRRQRR-KFERQKILD- YWTGS - C (SEQ ID NO:65)

N - KFERQKILD-GRKKRRQRR- YWTGS - C (SEQ ID NO: 66)

N - KFERQKILD- YWTGS GRKKRRQRR - C (SEQ ID NO: 67) In certain embodiments, the FRPs are encoded by mRNA. In some embodiments, the polynucleotides or mRNAs herein encoding the FRPs herein comprise at least one chemical modification or chemically modified base, nucleoside, or nucleotide. The chemical modifications may comprise any modification which is not naturally present in said RNA or any naturally-occurring modification of adenosine (A), guanosine (G), uridine (U), or cytidine (C) ribonucleosides. For example, a single polynucleotide or mRNA may include both naturally-occurring and non-naturally-occurring modifications. Chemical modifications may be located in any portion of the polynucleotide or mRNA molecule and the polynucleotide or mRNA molecule may contain any percentage of modified nucleosides (1- 100%, such as at least 20% ... at least 40% ... or at least 60%). In some embodiments, every particular base or nucleoside may be modified (e.g., every uridine is a modified uridine). In some embodiments, at least 20%, or 50%, or 80% of any single nucleotide (e.g., uracil) in the polynucleotide or mRNA is chemically modified. In some embodiments, a particular modification is used for every particular type of nucleoside or base (e.g., every uridine is modified to a 1-methyl-pseudouridine). Exemplary RNA modifications can be found in the RNA modification database (See, mods(dot)rna(dot)albany(dot)edu/home).

In some embodiments, the at least one chemical modification comprises a modified uridine residue. Exemplary modified uridine residues include, but are not limited to, pseudouridine, 1 -methylpseudouridine, 1 -ethylpseudouridine, 2-thiouridine, 4'- thiouridine,

5-methyluridine, 2-thio-l -methyl- 1 -deaza-pseudouridine, 2- thio-l-methyl-pseudouridine, 2- thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio- pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-l-methyl- pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-0-methyl uridine.

In some embodiments, the at least one chemical modification comprises a modified cytosine residue. Exemplary nucleosides having a modified cytosine include 5 -aza-cytidine,

6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-methyl-cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2- thio-cytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1 -methyl-pseudoisocytidine, 4-thio-l- methyl-l-deaza-pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza- zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy- cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1 -methyl- pseudoisocytidine, lysidine, a-thio-cytidine, 2'-O-methyl-cytidine, 5,2'-O-dimethyl-cytidine, N4-acetyl-2'-O-methyl-cytidine, N4,2'-O-dimethyl-cytidine, 5-formyl-2'-O-methyl-cytidine, N4,N4,2'-O-trimethyl-cytidine, 1 -thio-cytidine, 2'-F-aracytidine, 2'-F-cytidine, and 2'-0H- aracytidine.

In some embodiments, the at least one chemical modification comprises a modified adenine residue. Exemplary nucleosides having a modified adenine include 2- amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido- adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza- 2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl- adenosine, 2-methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6- isopentenyl-adenosine, 2-methylthio-N6-isopentenyl-adenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6- glycinylcarbamoyl-adenosine, N6-threonylcarbamoyl-adenosine, N6-methyl-N6- threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6- dimethyl-adenosine, N6-hydroxynoryalylcarbamoyl-adenosine, 2-methylthio-N6- hydroxynoryalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2-methylthio- adenine, 2-methoxy-adenine, a-thio-adenosine, 2'-O-methyl-adenosine, N6,2'-O-dimethyl- adenosine, N6,N6,2'-O-trimethyl-adenosine, l,2'-O-dimethyl-adenosine, 2'-O- ribosyladenosine (phosphate), 2-amino-N6-methyl-purine, 1 -thio-adenosine, 8-azido- adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino- pentaoxanonadecyl)-adenosine.

In some embodiments, the at least one chemical modification comprises a modified guanine residue. Exemplary nucleosides having a modified guanine include inosine, 1 - methyl-inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wybutosine, peroxy wybutosine, hydroxywybutosine, undermodified hydroxywybutosine, 7-deaza- guanosine, queuosine, epoxy queuosine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano- 7-deaza-guanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza- guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7- methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1- methyl-guanosine, N2-methyl-guanosine, N2,N2-dimethyl-guanosine, N2,7-dimethyl- guanosine, N2,N2,7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1- methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a- thio-guanosine, 2'-O-methyl-guanosine, N2-methyl-2'-O-methyl-guanosine, N2,N2-dimethyl- 2'-O-methyl-guanosine, l-methyl-2'-O-methyl-guanosine, N2,7-dimethyl-2'-O-methyl- guanosine, 2'-O-methyl-inosine, l,2'-O-dimethyl-inosine, and 2'-O-ribosylguanosine (phosphate). In certain embodiments, the nucleic acid sequences or mRNAs encoding the FRPs described herein are present in lipid nanoparticles (e.g., for intravenous delivery to a human). Lipid nanoparticle compositions may include one or more cationic and/or ionizable lipids, phospholipids, neutral or non-cationic lipids, polyethylene glycol (PEG)-lipid conjugates, and/or sterols. In some embodiments, the lipid nanoparticle comprises a cationic lipid and/or ionizable lipid, a neutral or non-cationic lipid, and cholesterol. Cationic and/or ionizable lipids include, for example, amine-containing lipids that can be readily protonated and may have a positive or partial positive charge at physiological pH due to a pKa value between pH 5 and 8. The polar headgroup of the cationic lipids preferably comprises amine derivatives such as primary, secondary, and/or tertiary amines, quaternary ammonium, various combinations of amines, amidinium salts, or guanidine and/or imidazole groups as well as pyridinium, piperazine and amino acid headgroups such as lysine, arginine, ornithine and/or tryptophan. Cationic lipids include, but are not limited to, l,2-dimyristoyl-sn-glycero-3- ethylphosphocholine (DMEPC), l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or l,2-dioleoyl-3-trimethylammonium propane (DOTAP), l,2-dimyristoyl-3- trimethylammonium propane (DMTAP), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)- dimethylazanium bromide (DMRIE), didodecyl(dimethyl)ammonium bromide (DDAB), 1 ,2- dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 3|3-[N — (N\N'- dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol) or dioleyl ether phosphatidylcholine (DOEPC). Ionizable lipids include, but are not limited to, l,2-dioleyloxy-3-dimethylamino- propane (DODMA).

In some embodiments, the lipid nanoparticle comprises a polyethylene glycol (PEG)- lipid conjugate. A PEG-lipid conjugate may include, but is not limited to, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacyl glycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-DMG (1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol), PEG-c-DOMG (R-3-[(oj-methoxy poly(ethylene glycol)2000)carbamoyl)]-l,2-dimyristyloxlpropyl-3-amine), PEG-DMA (PEG- dimethacrylate), PEG-DLPE ( 1 ,2-didodecanoyl-sn-glycero-3-phosphoethanolamine-PEG), PEG-DMPE (PEG- l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), PEG-DPPC (PEG- dipalmitoyl phosphatidylcholine), PEG-N,N-di(tetradecyl)acetamide, or a PEG-DSPE (1, 2- distearoyl-sn-glycero-3 -phosphoethanolamine-poly (ethylene glycol)) lipid. In some embodiments, the lipid nanoparticle comprises PEG-DMG and/or PEG-N,N- di(tetradecyl)acetamide. The sterol may comprise cholesterol, fecosterol, ergosterol, campesterol, sitosterol, stigmasterol, brassicasterol or a sterol ester, such as cholesteryl hemisuccinate, cholesteryl sulfate, or any other derivatives of cholesterol. A neutral or non-cationic lipid may include one or more phospholipids. Phospholipids include a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety may include, but is not limited to, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin. A fatty acid moiety may include, but is not limited to, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

Phospholipids suitable for use in the compositions may include, but are not limited to, phosphatidylglycerol (PG) including dimyristoyl phosphatidylglycerol (DMPG) and 1 ,2- dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG); phosphatidylcholine (PC), including egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di- O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl-sn- glycero- 3 -phosphocholine (Cl 6 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diarachidonoyl-sn-glycero-3- phosphocholine; phosphatidylethanolamine (PE) including l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine; phosphatidic acid (PA); phosphatidylinositol (PI); phosphatidylserine (PS); and sphingomyelin (SM).

The positively charged lipid structures described herein may also include other components typically used in the formation of vesicles (e.g., for stabilization). Examples of such other components includes, without being limited thereto, fatty alcohols, fatty acids, and/or any other pharmaceutically acceptable excipients which may affect the surface charge, the membrane fluidity and assist in the incorporation of the lipid into the lipid assembly. EXAMPLES

EXAMPLE 1

To pursue an opioid-alternative and promising therapeutic approach to treat chronic neuropathic pain, we have tested an embodiment of an innovative peptide, called fibronectin reduction peptide (FRP). One embodiment of FRP, tested in this example, has a sequence shown in SEQ ID NO:4 and is 35 amino acids in length and is composed of three unique motifs, including an N-terminal cell membrane, and blood brain barrier, penetrating motif (SEQ ID NO: 1), a central fibronectin binding motif (SEQ ID NO:2), and a C-terminal lysosome targeting motif (SEQ ID NO:3). These elements conceptually allow FRP to: (1) efficiently distribute into the nervous system via its N-terminal motif; (2) specifically bind to fibronectin via its fibronectin binding motif; and (3) degrade fibronectin-FRP complexes in the lysosome via its C-terminal motif (see Figure 1). These elements are important as FRPs are translatable and specific approach to target fibronectin/integrin pathways that are important for inflammatory, fibrotic and/or nociceptive responses.

In this Example, we have conducted experiments to validate the therapeutic efficacy of this novel FRP approach in rats with chronic constriction injury (CCI)⁷, a widely used pre- clinical model of chronic neuropathic pain, using behavioral assessments and histological studies (Figure 2). We have observed significantly-increased fibronectin levels in the injured sciatic nerve and the dorsal root ganglion (DRG) of rats with CCI (Figures 4, 5), which are associated with clearly-developed allodynia and hyperalgesia (Figures 6, 7, 8), the classic symptoms of chronic pain.

FRP for the management of pain should substantially decrease the use of opioids and subsequent OUD, which will lead to FRP as a safe, non-addictive analgesic to replace opioids as the first-line treatment for moderate to severe pain. The sequence details of the particular FRP tested in this example are below.

In certain embodiments, the FRP has the following sequence:

N - YGRKKRRQRRR-GAPGSSYWTG-KFERQKILDQRFFE - C (SEQ ID NO:4).

The SEQ ID NO:4 sequence has the following three components:

A fibronectin binding motif: GAPGSSYWTG (SEQ ID NO:2); and A lysosome targeting motif: KFERQKILDQRFFE (SEQ ID NOG). The data generated shows that injected FRP is distributed in the lesion epicenter and in areas both rostral and caudal to it in sciatic nerve. One of the first questions to address is whether FRP can diffuse into the injured rat sciatic nerve when injected subcutaneously. Fluorescein isothiocyanate (FITC)-conjugated FRP (FFTC-FRP) was subcutaneously injected near the injury site once daily for 7 days beginning 1 week after CCI in order to investigate its distribution in the sciatic nerve. The results revealed extensive distribution of the injected FITC-FRP within the sciatic nerve, especially around the injury epicenter (Figure 3). These data support the concept that FRP is able to reach the injury site.

The second important data show that FRP treatment significantly decreased CCI- upregulated fibronectin in both sciatic nerve and DRG at 3 weeks after CCI. We examined the pathological expression levels of fibronectin in sciatic nerves (Figure 4) and DRG (Figure 5) after CCI. Immunohistochemical staining showed that fibronectin levels were significantly induced by CCI when compared to uninjured groups and continuously increased at 3 weeks after CCI (Figures 4, 5). Importantly, one-week delayed FRP treatment, composed of daily subcutaneous injections beginning one week after CCI and lasting for two additional weeks, significantly decreased CCI-upregulated fibronectin when compared to scrambled peptide treatment. These data support the hypothesis that (1) fibronectin is pathologically upregulated after injury, indicating the roles that fibronectin plays in the pathogenesis, including development of chronic neuropathic pain after CCI; and (2) FRP can significantly decrease pathologically-upregulated fibronectin.

The third data demonstrate that one-week delayed FRP treatment can significantly alleviate chronic neuropathic pain follow CCI. We tested whether long-term systemic treatment with FRP that targets fibronectin would be capable of improving the long-term behavioral outcome of rats following chronic CCI. The animals received 14 consecutive daily subcutaneous injections of either FRP or scrambled peptide into the leg near the lesion, with injections starting one week after CCI. The time point (seven days after CCI) was the time when chronic neuropathic pain developed in injured hindpaw when compared with noninjured hindpaw. The symptoms of chronic neuropathic pain are allodynia, a persistent sensitivity to touch or temperature, and hyperalgesia, a disabling pain caused by low levels of stimuli that normally go unnoticed in a healthy person¹. The pain-associated behaviors including thermal hyperalgesia (Figure 6), mechanical allodynia (Figure 7) and mechanical hyperalgesia (Figure 8) were assessed. One-week delayed FRP treatment clearly decreased hindpaw thermal hyperalgesia compared to the scrambled peptide-treated group when receiving noxious heat (Figure 6). In addition, one-week delayed FRP treatment can significantly improve CCI-induced mechanical allodynia by increasing both the force and latency needed for hindpaw withdrawal, as compared to scrambled peptide- treated animals when receiving innocuous touch (Figure 7). Similarly, one-week delayed FRP treatment can significantly decrease CCI-induced mechanical hyperalgesia by increasing both the force and latency needed for hindpaw withdrawal, as compared to scrambled peptide-treated animals when receiving noxious touch (Figure 8). These data demonstrate the promising potential to develop FRP as a safe, non-addictive analgesic for chronic pain treatment.

REFERENCES:

1. Descalzi, G. et al. Epigenetic mechanisms of chronic pain. Trends Neurosci 38, 237- 246 (2015).

2. Dahlhamer, J. et al. Prevalence of Chronic Pain and High-Impact Chronic Pain Among Adults - United States, 2016. MMWR Morb Mortal Wkly Rep 67, 1001-1006 (2018).

3. Boscarino, J. A. et al. Risk factors for drug dependence among out-patients on opioid therapy in a large US health-care system. Addiction 105, 1776-1782 (2010).

4. Huskamp, H. A. et al. Long-Term Prospects for Telemedicine in Opioid Use Disorder (OUD) Treatment: Results from a Longitudinal Survey of OUD Clinicians. I Gen Intern Med 38, 2139-2146 (2023).

5. Segel, J. E. & Winkelman, T. N. A. Persistence and Pervasiveness : Early Wave Opioid Overdose Death Rates Associated With Subsequent Overdose Death Rates. Public Health Rep 136, 212-218 (2021).

6 McCance-Katz, E. F. The Substance Abuse and Mental Health Services Administration (SAMHSA): New Directions. Psychiatr Serv 69, 1046-1048 (2018).

7. Bennett, G. J. & Xie, Y. K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33, 87-107 (1988).

8 Campana, G. & Rimondini, R. Mechanical Nociception in Mice and Rats: Measurement with Automated von Frey Equipment. Methods Mol Biol 2201, 195-198 (2021).

9. Perez de Vega et al., Recent progress in non-opioid analgesic peptides. Arch Biochem Biophys 660, 36-52, 2018.

10. Fleming, J. C. et al. Alpha4betal integrin blockade after spinal cord injury decreases damage and improves neurological function. Exp Neurol 214, 147-159, 2008.

11. Lin et al., Fibronectin inhibits chronic pain development after spinal cord injury. Journal of neurotrauma 29, 589-599, 2012. 12. Liu et al. The alternatively spliced fibronectin CS1 isoform regulates IL-17A levels and mechanical allodynia after peripheral nerve injury. J Neuroinflammation 12, 158, 2015

13. W02019/005822A1

All publications and patents mentioned in the specification and/or listed below are herein incorporated by reference. V arious modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope described herein.

Claims

CLAIMS We Claim:

1. A method of treating fibronectin-related inflammation and/or fibrosis and/or neuropathic pain in a subject comprising: administering a fibronectin reduction peptide (FRP), or a nucleic acid sequence encoding said FRP, to a subject with fibronectin-related inflammation and/or fibrosis and/or neuropathic pain, wherein said FRP comprises: i) a cell membrane and blood brain barrier penetrating (CMBBBP) motif, ii) a fibronectin binding motif, and iii) a lysosome targeting motif.

2. The method of claim 1, wherein said subject has at least one of the following: pain, nervous system disorder, neurodegenerative disease, an organ transplant, lung fibrosis, liver fibrosis, an amputation, cosmetic surgery, or a plastic surgery application.

3. The method of claim 1, wherein said neuropathic pain is localized to at least one peripheral nerve of said subject.

4. The method of claim 3, wherein said administering reduces the level of fibronectin present at said at least one peripheral nerve.

5. The method of claim 1, wherein said administering is performed systemically, optionally in the area of a peripheral nerve of said subject,

6. The method of claim 1, wherein said administering is performed subcutaneously, optionally in the area of a peripheral nerve of said subject.

7. The method of claim 1, wherein said administering is repeated daily for at least 3 days, optionally near an injured nerve, and optionally for at least 10 days.

8. The method of claim 1, wherein said administering is conducted after at least two days of initiation of said fibronectin-related inflammation, fibrosis and/or neuropathic pain condition.

9. The method of claim 1 , wherein said administering is conducted after at least one weeks of initiation of said fibronectin-related inflammation, fibrosis and/or neuropathic pain condition.

10. The method of claim 1, wherein said CMBBBP motif is located at the N-terminal, C- terminal or central of said FRP.

11. The method of claim 1 , wherein said lysosome targeting motif is located at the N- terminal, C-terminal or central of said FRP.

12. The method of claim 1, wherein said fibronectin binding motif is located between the CMBBBP motif and the lysosome targeting motif or is located at the N-terminal or C- terminal of said FRP.

13. The method of claim 1, wherein said CMBBBP motif comprises or consists of an amino acid sequence shown in SEQ ID NOS: 1 or 5-27, or a sequence with one or two amino acid additions, subtractions, or substitutions.

14. The method of claim 1, wherein said fibronectin binding motif comprises or consists of an amino acid sequence shown in SEQ ID NOS:2 or 28-47, or a sequence with one or two amino acid additions, subtractions, or substitutions.

15. The method of claim 1, wherein said lysosome targeting motif comprises or consists of an amino acid sequence shown in SEQ ID NO:3 or 48-56, or a sequence with one or two amino acid additions, subtractions, or substitutions.

16. The method of claim 1 , wherein said FRP comprises or consists of an amino acid sequence shown in SEQ ID NO:4 or 57-67, or a sequence with one or two amino acid additions, subtractions, or substitutions.

17. A composition, system, or kit comprising: a fibronectin reduction peptide (FRP) of any of claims 1-16, or a nucleic acid sequence encoding said FRP.

18. The composition, system, or kit of claim 17, further comprising a device for injecting a composition comprising said FRP into a subject’s peripheral nervous system, optionally, wherein said injecting is subcutaneous.