Patent Description:
There is a huge market desire to remove scares caused by trauma and/or neurodegeneration to facilitate nerve regrowth and to enhance functional outcomes in both Peripheral Nervous System (PNS) and Central Nervous System (CNS) clinically. There is currently no intervention available clinically to target the spinal cord injury (SCI) and to address its associated neurological disorders including bladder control. The patients with SCI in the U. have been estimated to be approximately <NUM>,<NUM> people, with a range from <NUM>,<NUM> to <NUM>,<NUM> persons in <NUM>. The new cases are about <NUM>,<NUM> per year. The bladder function is a high priority that patients with SCI would like to be returned.

There are two major approaches applied in experiments to reduce chondroitin sulfate proteoglycan (CSPG, the major component of scarring) are bacterial enzyme chondroitinase ABC and Lentiviral delivery of chondroitinase ABC. However, both chondroitinase ABC and Lentiviral delivery of chondroitinase ABC have only limited efficacy and are not clinically applicable. Chondroitinase ABC has low thermal stability, short longevity and must be applied locally. These disadvantages limit its efficacy and clinical application. Lentiviral delivery of chondroitinase ABC has a relatively low transfection rate, and biological safety concerns. In addition, Lentivirus needs to be applied about two weeks before injury/trauma. The timing and amount applied cannot be controlled. All of these disadvantages limit efficacy and clinical application of Lentiviral delivery of chondroitinase ABC.

Another newly published peptide, intracellular sigma peptide (ISP), was originally designed to interfere the function of one of the receptors of CSPG (i.e. PTPσ) in order to block the inhibitory effects of CSPG, but has no effects on CSPG itself in vivo. While ISP when applied immediately after SCI in rats has been reported to have minor effects on promoting functional recovery after SCI, the underlying mechanisms are still unknown even after extensive investigation for years. The most critical disadvantage of ISP is that ISP has no effects on chronic SCI when ISP was applied two-month after SCI. Such disadvantages and the unknown mechanisms underlying its published effects significantly limit the efficacy and clinical application of ISP.

<CIT> teaches a system to knock down an endogenous protein by using the endogenous lysosome-dependent autophagy system, chaperone mediated autophagy. Said prior art discloses the concept of making a tripartite fusion protein comprising a cell membrane penetrating domain, a specific protein binding domain and a lysosome targeting domain as well as a medical use of said fusion polypeptide for the treatment of neurodegenerative diseases. However, <CIT> fails to disclose a fusion protein comprising a chondroitin sulfate proteoglycan (CSPG) binding domain, which distinguishes the claimed subject-matter over said prior art. While there exist in-vitro data disclosure in the prior art, see e.g., <NPL>, showing that PTPσ has a binding site for CSPG, said disclosure does not go beyond what has already been discussed in the above background section of the present application with regard to ISP (intracellular sigma peptide). As outlined in in the preceding paragraph, in-vitro data assumptions cannot be extrapolated to the clinical (in-vivo) situation, which is completely different and has been shown to fail.

Provided herein are compositions, systems, kits, and compositions, systems, kits for use in methods of promoting motor function recovery, and/or bladder function recovery, after spinal cord injury in a subject as defined in the claims.

The reference to methods of treatment by therapy or surgery or in vivo diagnosis methods in the examples of this description are to be interpreted as references to compounds, pharmaceutical compositions and medicaments of the present invention for use in those methods. Therefore, for illustration only, provided herein are methods of treating nervous system injury or trauma or degeneration or aging (e.g., advanced years, such as over <NUM> years old) in a subject comprising: administering a CSPG reduction peptide (CRP), or a nucleic acid sequence encoding the CRP, to a subject with a nervous system injury or trauma or degeneration or aging, wherein the CRP comprises: i) a first amino acid sequence encoding a cell membrane penetrating domain, ii) a second amino acid sequence encoding a chondroitin sulfate proteoglycan (CSPG) binding domain, and iii) a third amino acid sequence encoding a lysosome targeting domain.

The nervous system injury or trauma or degeneration or aging may be localized to at least one nervous system site on the subject (e.g., particular location between certain vertebrate on the spinal cord). The administering may be under conditions such that the CRP reduces the level of CSPG present at the at least one nervous system site (e.g., one site, two sites, three sites, etc.). The nervous system site may be in the spinal cord of the subject. The nervous system site may be in the degenerated brain and/or spinal cord of the subject. the nervous system injury or trauma or degeneration or aging may be to the subject's central nervous system (CNS). The nervous system injury or trauma or degeneration or aging may be to the subject's peripheral nervous system (PNS).

Administering may be conducted within about <NUM> hours of the nervous system injury or trauma or degeneration or aging (e.g., within <NUM> hour. <NUM> hours. <NUM> hours. <NUM> hours, or within <NUM> hours). The administering may be conducted after at least two days of the nervous system injury or trauma or degeneration or aging (e.g., after <NUM> days. <NUM> days. or <NUM> days). the administering may be conducted at least one week after the nervous system injury or trauma or degeneration or aging (e.g., after <NUM> days. <NUM> days. or <NUM> days). The administering may be conducted at least one month after the nervous system injury or trauma or degeneration or aging (e.g., at least one month or <NUM> months). The administering may be conducted at least two months after nervous system injury or trauma or degeneration or aging (e.g., at least <NUM> months. <NUM> months. <NUM> year. <NUM> years. or <NUM> years after the injury or trauma).

For illustration only, provided herein are methods of treating multiple sclerosis (MS), or aiding in limb transplant, in a subject comprising: administering a CSPG reduction peptide (CRP), or a nucleic acid sequence encoding the CRP, to a subject with MS (or other neurological condition) or undergoing limb transplant, wherein the CRP comprises: i) a first amino acid sequence encoding a cell membrane penetrating domain, ii) a second amino acid sequence encoding a chondroitin sulfate proteoglycan (CSPG) binding domain, and iii) a third amino acid sequence encoding a lysosome targeting domain.

Provided herein are compositions, systems, and kits as defined in claim <NUM> and claim <NUM> In particular embodiments, the compositions, systems, further comprise a device for injecting a composition comprising the CRP into a subject's nervous system as defined in claim <NUM>. Further provided herein are compositions, systems, or kits of claim <NUM> or claim <NUM> for use in a method of promoting motor function recovery, and/or bladder function recovery, after spinal cord injury in a subject as defined in claims <NUM> and <NUM>.

Provided herein are compositions, systems, kits, and compositions, systems, kits for use in methods of promoting motor function recovery, and/or bladder function recovery, after spinal cord injury in a subject as defined in the claims.

Chondroitin sulfate proteoglycans (CSPGs) are the major components of scar, which are significantly increased after injury/trauma to both peripheral nervous system (PNS) and central nervous system (CNS). However, there is no clinically applicable strategy to reduce CSPGs now. The CSPG reduction peptides (CRPs), and nucleic acid sequences encoding CRPs, provide an effective way to remove CSPGs from the site of injury caused by trauma or neurodegeneration or aging to CNS and PNS. The benefits of removing CSPGs after injury include promoting the regrowth of damaged nerves and enhancing functional outcomes.

As defined in claim <NUM>, the CRP may comprise the following sequence: N - YGRKKRRQRRR-PKPRVTWNKKGKKVNSQRF-KFERQKILDQRFFE - C (SEQ ID NO:<NUM>). 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:<NUM>. The SEQ ID NO:<NUM> sequence has the following three components:.

Each of these domains may be constructed with longer, shorter, or mutated versions of the sequences shown in SEQ ID NOS: <NUM>-<NUM> and <NUM>. For example, one could change one, two, three 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 similar side chains. 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. Provided herein are peptides that have substantial identity to at least a portion of the amino acid sequences shown in SEQ ID NOs: <NUM>-<NUM>.

The N-terminal cell-membrane penetrating domain of a CRP according to the invention is defined in claim <NUM>, but may be also an amino acid sequence as shown in SEQ ID NOs: <NUM> and <NUM>-<NUM> of Table <NUM> 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 <NUM>.

The cell membrane penetrating peptide according to the invention is defined in claim <NUM>, but may be also a protein transduction domain (PTD) with the presence of multiple arginine (R) residues as shown in SEQ ID NOs: <NUM> and <NUM>-<NUM> of Table <NUM> or a cell penetrating peptide (CPP) from the CPPsite <NUM>, which is an updated version of database CPPsite. This site contains around <NUM> unique cell penetrating peptides (CPPs) along with their secondary & tertiary structure, and can be found at www. followed by "crdd. net/raghava/cppsite/.

The C-terminal lysosomal targeting domain of a CRP is defined in claim <NUM>, but may be also an amino acid sequence as shown in SEQ ID NOs: <NUM> and <NUM>-<NUM> of Table <NUM> or such a peptide with one, two, or three (or more) N-terminal or C-terminal additions, subtractions or mutations therein or the lysosomal targeting domain is a chaperone-mediated autophagy (CMA)-targeting motif (CTM) containing a KFERQ-like motif, such as those shown in SEQ ID NOs: <NUM> and <NUM>-<NUM> of Table <NUM>. 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 <NUM>.

In certain embodiments, the CSPG binding domain of a CRP is defined in claim <NUM>, but may be also an amino acid sequence as shown in SEQ ID NOs: <NUM> and <NUM>-<NUM> of Table <NUM> 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 <NUM>.

This Example describes treating spinal cord injury (SCI) in a model using a CSPG reduction peptide (CRP). The CRP had the following three components:.

The full CRP sequence is as follows:
N - YGRKKRRQRRR-PKPRVTWNKKGKKVNSQRF-KFERQKILDQRFFE - C (SEQ ID NO:<NUM>).

The designed CRP (SEQ ID NO:<NUM>) includes an N-terminal cell membrane-penetrating domain (SEQ ID NO: <NUM>), a central CSPGs binding domain (SEQ ID NO:<NUM>), and a C-terminal lysosome targeting domain (SEQ ID NO:<NUM>) for directing the CRP-CSPGs complex to lysosomes for degradation. <FIG> shows a schematic of the exemplary CRP employed in this Example, as well as some of the benefits of such constructs.

In this Example, the CRP has been applied in a preclinical rat model to test the efficacy of the treatment of spinal cord injury (SCI). The injury was made at thoracic <NUM> level using contusive injury device. The contusive SCI is the most clinical relevance among all experimental SCI models. We have assessed both motor and bladder control as functional outcomes and analyzed anatomical evidences to support these findings. We also selected sub-acute phase (ONE-DAY after SCI) and chronic phase (TWO-MONTHs after SCI) to start our treatment. <FIG> shows a schematic of the experimental design for the in vivo testing in this Example.

For the sub-acute treatment, continuous daily subcutaneous injection of CRP beginning one-day after SCI significantly reduces SCI-induced overexpression of CSPGs. Furthermore CRP can efficiently improve locomotion and bladder electromyography (EMG) activities and voiding patterns after SCI. Particularly, we found that CRP increases sprouting/regeneration of axons including serotonin (<NUM>-HT) and anterogradely traced fibers from brain stem and red nuclei, the critical pathways regulating both locomotion and bladder functions, below the SCI lesion and even in the lumbosacral spinal cord.

The results of this testing are shown in the figures. In particular, <FIG> shows histological results from the sub-acute treatment, where CRP effectively decreased CSPG in lesion epicenter and adjacent tissue two weeks after SCI. <FIG> shows results from the sub-acute treatment, where CRP significantly decreased CSPG three months after SCI. <FIG> shows results out to <NUM> days post-contusion shows that CRP significantly promotes motor function recovery after SCI. <FIG> shows that CRP improves voiding patterns after SCI. <FIG> shows that CRP improves bladder function after SCI, reducing residual volume and volume per void toward normal condition. <FIG> shows that CRP improves bladder function after SCI, reducing overactive bladder by the reduction of both the number and amplitude of non-voiding contraction. <FIG> shows that CRP improves bladder function after SCI, improving external urethral sphincter (EUS) activity by increasing the number of burst, the EMG amplitude, and the coordination with detrusor contraction during the void period. <FIG> shows CRP enhances sprouting of serotonin (<NUM>-HT) fibers below lesion after SCI. <FIG> shows that CRP enhances nerve sprouting below lesion from neuron in brain stem and red nucleic after SCI.

The above were followed up with studies to evaluate the treatment effects of CRP in chronic SCI. The chronic SCI is not only way more challenge to be repaired but also is the clinical status of the majority of patients with SCI. Our results find that CRP subcutaneously injected even beginning two-month after SCI still significantly improves the locomotion and voiding patterns.

In addition, we made a comparison of CRP with recently developed peptides, intracellular sigma peptide (ISP) targeting CSPG receptor pathways. Our data showed (<NUM>) CRP but not ISP could reduce CSPG after SCI and (<NUM>) importantly, CRP but not ISP could improve functional outcomes after chronic phase of SCI. <FIG> shows CRP, but not ISP, effectively decreases CSPG in lesion epicenter and adjacent tissue two weeks after SCI. <FIG> shows that CRP, but not ISP, when applied two months after SCI effectively promoted locomotion recovery.

Because CSPGs are the major physical barriers for axonal regeneration and functional recovery after trauma in both PNS and CNS and after neurodegenerative disorders, CRP type construction could apply to many disorders other than SCI. Indeed, CRP could be applied to limb transplants. In addition, neurodegeneration-induced increases in heparin sulfate proteoglycans (HSPGs) as well as CSPGs are unbeatable barriers for current treatment of Multiple Sclerosis (MS), which indicates the therapeutic potential of CRP to treat neurodegenerative disorders such as MS and related conditions.

This Example describes additional in vitro and in vivo work with the CSPG reduction peptide (CRP) described in Example <NUM>.

<FIG> shows that applied CRP is found in the lysosome of Neu7 astrocytes and provides the results of an in vitro assays that demonstrates the concept of lysosmoal target domain in CRP. Such an in vitro assay was used to demonstrate the co-localization of FITC-CRP and lysosomal marker (LAMP1). Representative images indicate that FITC-CRP is brought to lysosomes (LAMP1+) for degradation after entering Neu7 cells (arrow).

<FIG> shows that CRP significantly decreases Neu7-produced CSPGs. In particular, <FIG> shows that CRP significantly reduces CSPG produced by Neu7 cells to a comparable level what ChABC does. Note there is also a dosage response by CRP application.

<FIG> shows that CRP treatment improves gait patterns after SCI in rats. In particular <FIG> shows Kinematics assessment in SCI rats. (A) Representative total of hindlimb movement trajectories for spinal-intact, SCI+scrambled peptide (scr) and SCI+CRP groups at <NUM> months post-SCI. (B-G) CRP treatment significantly improved several parameters of gait pattern with trajectories more consistent to what the spinal-intact animals did, when compared to scr treatment. *, P< <NUM>; ***, P< <NUM> when compared to the spinal-intact group; +, P< <NUM>; +++, P< <NUM> when compared to scr group. N=<NUM> to <NUM> per group.

<FIG> shows that CRP treatment improves bladder function after SCI. (A) Representative cystometrograms (CMG) with micturition (voiding) events (indicated by asterisk) for spinal-intact, SCI+scrambled peptide (scr) and SCI +CRP groups at <NUM> months post-SCI. (B~H) CRP treatment reduces hyperactive bladder and improve CMG parameters including lower voiding pressure and smaller bladder capacity when compared to scr application after T8 SCI. *, P< <NUM>; **, P< <NUM>; ***, P< <NUM> when compared to the spinal-intact group; +++, P< <NUM> when compared to the scr group. N=<NUM> to <NUM> per group.

<FIG> shows that CRP treatment improves EUS EMG activity during the void after SCI (more bursting number and larger bursting amplitude). (A) Representative cystometrograms (CMG) with external urethral sphincter (EUS) electromyogram (EMG) recordings at micturition events for spinal-intact, SCI+scrambled peptide (scr) and SCI+CRP groups at <NUM> months post-SCI. (B-D) CRP treatment improves EUS bursting activity when compared to scr application after T8 SCI. The red dot indicates bursting activity. **, P< <NUM>; ***, P< <NUM> when compared to the spinal-intact group; +, P< <NUM>, + +, P< <NUM>; +++, P< <NUM> when compared to the scr group. N=<NUM> to <NUM> per group.

<FIG> shows that CRP treatment reduced PNN in lumbosacral spinal cord after T8 SCI, and particularly shows the anatomical assessments of perineuronal nets (PNN) in SCI rats. (A, B) Representative L4 spinal cord transverse images show CRP treatment reduces overall PNN in grey matter of lumbar spinal cord neurons at <NUM> months post SCI. Notice areas with clear decreased WFA staining of the PNN (asterisk). (C, D) Statistical analyses show the significant reduction of PNN at both L4 and L6/S1 levels in CRP group when compared to the scrambled peptide group. (E, F) Representative higher power immunostaining images of the ventral horn of transverse L4 sections show the strong PNN intensity (arrow) surrounding the NeuN+ cells in scr treated SCI rats (E), while CRP treatment reduces them (F). *, P< <NUM> when compared to the scr treatment. N=<NUM> per group.

<FIG> shows that CRP enhances serotonergic fibers sprouting in lumbrosacral spinal cord. In particular, 5HT immunoreactivity in lumbosacral level after SCI. (A-F) Representative spinal cord transverse images show that pretty low amount of 5HT fibers (arrow) found at both L4 (A-C) and L6/S1 (D-F) levels after severe contusive SCI, while CRP treatment significantly increases immunoreactivity of 5HT fibers. (G, H) Representative images with double staining of WFA and 5HT showing areas of clear decreased PNN indicated by WFA+, in areas of intense <NUM>-HT sprouting (asterisk). ***, P< <NUM> when compared to the scrambled peptide treatment. N= <NUM> per group.

<FIG> shows that CRP improves locomotion after chronic SCI. In particular, <FIG> shows BBB scores that show that CRP treatment improves hindlimb locomotion after chronic SCI (N=<NUM> to <NUM> animals per group). Note the significant increases in BBB locomotion scale by CRP at <NUM> months post SCI when compared to what at the time (two months post SCI) of beginning treatment.

<FIG> shows that CRP treatment improves gait patterns after chronic SCI. In particular, <FIG> shows representative 2D trajectory patterns indicated CRP treatment improved stepping kinematic of both hindlimbs by CRP at <NUM> months post SCI when it compared to what at the time (two months post SCI) of beginning treatment.

<FIG> shows that CRP treatment improves bladder function after chronic SCI. In particular, <FIG> shows representative cystometrograms (CMG) data indicated <NUM> months of CRP treatment (starting at <NUM> months post SCI) can reduce hyperactive bladder and improve other CMG parameters.

<FIG> shows that CRP treatment improves EUS EMG activity during the void after chronic SCI. In particular, <FIG> shows representative urodynamic and external urethral sphincter (EUS) electromyogram (EMG) recordings indicated CRP treatment can improve EUS bursting activity during the void period when compared to the scrambled peptide application after chronic SCI. The box area indicates the bursting activity.

<FIG> shows CRP enhances 5HT fibers sprouting in lumbar cord after chronic SCI. In particular, <FIG> shows representative spinal cord transverse images that CRP treatment can enhance 5HT+ nerve fibers sprouting in lumbosacral level when compared to the scrambled peptide treatment after chronic SCI.

<FIG> shows CRP treatment enhances 5HT sprouting but decreases PNN in lumbar cord after chronic SCI. In particular, <FIG> shows 5HT immunoreactivity in lumbosacral level after chronic SCI. Representative spinal cord transverse images show that low amount of 5HT fibers found at L4 levels after contusive SCI, while CRP treatment significantly increases immunoreactivity of 5HT fibers. Representative images with double staining of WFA and 5HT shows areas with robustly decreased PNN (indicated by WFA+) but enhanced <NUM>-HT sprouting.

Claim 1:
A composition, system, or kit comprising: a CSPG reduction peptide (CRP), or a nucleic acid sequence encoding said CRP, wherein said CRP is a fusion protein comprising the following:
a) a first amino acid sequence encoding a cell membrane penetrating domain, wherein said first amino acid sequence comprises SEQ ID NO: <NUM>;
b) a second amino acid sequence encoding a chondroitin sulfate proteoglycan (CSPG) binding domain, wherein said second amino acid sequence comprises SEQ ID NO:<NUM>; and
c) a third amino acid sequence encoding a lysosome targeting domain, wherein said third amino acid sequence comprises SEQ ID NO:<NUM>.