CN117677630A - Granulin/Epithelin modules and combinations thereof for the treatment of neurodegenerative diseases - Google Patents

Abstract

The present invention relates to methods and compositions comprising granulin/epithelin modules (GEMs) or combinations thereof suitable for the treatment of neurodegenerative diseases. The invention also relates to methods of treating neurodegenerative diseases, such as methods of administering therapeutic recombinant GEM proteins or gene therapy for delivering recombinant GEM gene products.

Description

Granulin/epithelialin modules and combinations thereof for the treatment of neurodegenerative diseases

Technical Field

The present invention belongs to the field of drugs and biological preparations for treating brain diseases. The present invention relates to granulin/epithelialin modules (GEMs) or combinations thereof. The present invention relates to methods and compositions comprising granulin/epithelialin modules (GEMs) or combinations thereof suitable for treating neurodegenerative diseases.

The present invention also relates to methods of treating neurodegenerative diseases using granulin/epithelial modules (GEMs) or combinations thereof, such as methods of administering therapeutic recombinant GEM proteins or gene therapy for delivering recombinant GEM gene products.

In an embodiment, the present invention relates to a recombinant polypeptide or a combination of multiple recombinant polypeptides, which comprises two to six granulin/epithelialin modules (GEMs). In an embodiment, the recombinant polypeptide or combination of polypeptides comprises two to six granulin/epithelialin modules (GEMs), including GEM E, and additionally one or more of GEM F, GEM C and GEM D.

In an embodiment, the invention relates to a therapeutic nucleic acid molecule or a combination of multiple therapeutic nucleic acid molecules expressing two to six granulin/epithelialin modules (GEMs). In an embodiment, the combination of granulin/epithelialin modules (GEMs) comprises two to six GEMs, including GEM E, and additionally one or more of GEM F, GEM C and GEM D.

Background Art

Progranulin (PGRN) is a growth factor-like protein that is involved in the regulation of multiple processes, including development, wound healing, angiogenesis, neuronal cell growth and maintenance, and inflammation. Altered PGRN expression has been demonstrated in a variety of neurodegenerative diseases, including Creutzfeldt-Jakob disease, motor neuron disease, Parkinson's disease, and Alzheimer's disease. For example, recent studies on the genetic etiology of neurodegenerative diseases have shown that inherited mutations in the PGRN gene may contribute to the adult onset of neurodegenerative diseases due to reduced neuronal survival.

Progranulin is associated with a variety of medical conditions. For example, PGRN is associated with lung inflammation, and PGRN is a known factor associated with Gaucher disease (a common lysosomal storage disease). PGRN is also associated with neuronal ceroid lipofuscinosis (NCL), which is also a lysosomal storage disease. Frontotemporal dementia is also associated with PGRN, and some FTDs are caused by mutations in GRN (the gene encoding PGRN) alleles. Spinal muscular atrophy is also associated with PGRN, where PGRN overexpression has been shown to reverse impaired development of primary motor neurons. PGRN also plays a role in ALS and Huntington's disease, and low PGRN appears to be a risk factor for ALS, PD, AD, schizophrenia and bipolar disorder. It is also known that low PGRN levels are associated with peripheral inflammatory conditions such as arthritis and atherosclerosis.

PGRN itself is a proprotein that can be cleaved into smaller domains, called granulin/epithelialin modules (GEMs), also known as granulin (A-G and p). Granulin has a highly disulfide-rich, evolutionarily conserved β-sheet fold. These properties are usually found in highly stable proteins that can withstand heat and pH changes. In fact, recent studies have emphasized that the cleavage of progranulin and therefore the generation of GEMs occurs in the acidic environment of lysosomes through the action of lysosomal cathepsins. Single GEMs may antagonize the functions of full-length proteins in cell growth and inflammation. Alternatively, some evidence suggests that the GEM domain also binds and stimulates cathepsin D (CTSD) enzyme-promoting activity. Although multi-GEM-sized peptides have been reported in highly degenerated brain regions of patients with Alzheimer's disease (AD), the actual molecular functions of single GEMs, multi-GEM fragments and full-length proteins are still not fully understood.

Currently, there is no effective treatment for many neurodegenerative diseases such as ALS, Alzheimer's disease or Parkinson's disease. Current treatments usually involve doctors trying to slow the progression of symptoms and make patients more comfortable. Although there are many drugs in development, the number of drugs approved by the FDA for treatment is limited (Riluzole for ALS; L-dopa for Parkinson's disease; cognitive enhancers, such as Aricept, for AD), and these treatments can only mask the progression of neurological diseases and may slightly prolong the lives of some patients.

Therefore, there is a great need for methods and compositions directed to treating neurodegenerative diseases.

Summary of the invention

In view of the prior art, the technical problem of the present invention is to provide alternative or improved agents suitable for the treatment of neurodegenerative diseases, in particular frontotemporal dementia (FTD), Parkinson's disease, Alzheimer's disease or amyotrophic lateral sclerosis. Another object of the present invention is to provide combinations of GEMs with improved biological properties relative to the aforementioned treatments.

This problem is solved by the features of the independent claim. Preferred embodiments of the invention are provided by the dependent claims.

Aspects and embodiments of the present invention are described as follows:

In one aspect, the present invention relates to a recombinant polypeptide or a combination of recombinant polypeptides comprising two to six granulin/epithelial modules (GEMs).

In one embodiment, the recombinant polypeptide comprises two to six granulin/epithelial modules (GEMs), including GEM E, and additionally one or more of GEM F, GEM B, GEM C, and GEM D.

In one embodiment, the recombinant polypeptide comprises two to six granulin/epithelial modules (GEMs), including GEM E, and additionally one or more of GEM F, GEM C, and GEM D.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) E, and additionally a GEMF.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) E, and additionally a GEMC.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) E, and additionally a GEMB.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) E, and additionally GEMF and GEM D.

In one embodiment, the recombinant polypeptide comprises granulin/epithelial module (GEM) E, and additionally GEMF, GEM D and GEM C.

In one embodiment, the recombinant polypeptide comprises two to six granulin/epithelial modules (GEMs), including GEM F, and additionally one or more of GEM A, GEM B, GEM E, GEM C, and GEM D.

In one embodiment, the recombinant polypeptide includes two to six granulin/epithelial modules (GEMs), including GEM F, and additionally one or more of GEM B, GEM E, GEMA, and GEM D. The combinations provided above have been shown to exhibit improved function using in vitro assessments relevant to determining therapeutic efficacy.

As shown in the Examples below, treatment with GEM A to F, optionally in combination, such as GEM E in combination with one or more of GEM A, GEM B, GEMF, GEM C and GEM D, showed improved cell survival levels of a motor neuron-like cell line (NSC-34) cultured with minimal serum.

To the best of the inventors' knowledge, the above combinations have not been previously disclosed or tested and represent an advantageous combination approach for treating diseases associated with neuronal cell death.

These combinations may be, but are not limited to, those derived from the studies in the examples disclosed below. For example:

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) F.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) B.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) C.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM)E.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) G.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM)A.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) D.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) F, and additionally GEMA.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) F, and additionally a GEMB.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) F, and additionally a GEMC.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) F, and additionally a GEME.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) F, and additionally a GEMD.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) B, and additionally a GEMC.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) B, and an additional GEME.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) C, and an additional GEME.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) E, and additionally GEMF and GEM B.

In one embodiment, the recombinant polypeptide comprises granulin/epithelial module (GEM) E, and additionally GEMF and GEM C.

In one embodiment, the recombinant polypeptide comprises granulin/epithelial module (GEM) E, and additionally GEMF, GEM D, GEM C and GEM B.

In one embodiment, the recombinant polypeptide comprises a granulin/epithelial module (GEM) E, and additionally a GEMF, no additional GEM, or one or more additional GEMs.

Surprisingly, these GEMs and GEM combinations were able to proliferate NSC34 cells to a greater extent than full-length progranulin. Improvements to full-length PGRN known in the art may be achieved by identifying individual GEMs or combinations of GEMs that exhibit beneficial effects on NSC34 cells.

Without being bound by theory, it appears that not all GEMs (as present in full-length PGRN) administered in combination are beneficial to NSC34 proliferation. When administered, it may be beneficial to isolate one or more GEMs rather than using the full combination of GEMs in full-length PGRN. Thus, the inventors have found that GEMs alone or in specific combinations can achieve improved effects over full-length PGRN, indicating that some GEMs or combinations of GEMs may represent a "version" or "truncated form" or "minimal form" of PGRN that is most suitable for neuronal cell proliferation.

In some embodiments, these GEMs or combinations of GEMs may be selectively effective against neuronal cells, or may induce proliferation of neuronal cells such as NSC34, but not other cell types. Thus, the inventors have identified optimal GEMs or combinations of GEMs that are "neuro-supportive," i.e., support neuronal survival and proliferation, and thus may be therapeutically beneficial in applications to reduce neuronal cell death, i.e., in the treatment of the diseases disclosed herein.

In embodiments, the GEMs or GEM combinations of the present invention are able to "reduce the complexity of active molecules" compared to full-length PGRN. Without being bound by theory, the inventors speculate that by reducing the full-length PGRN molecule to a selection of GEM combinations including 2-6 GEMs, potentially unnecessary or even toxic aspects of the full-length PGRN molecule can be removed, thereby improving the active agent compared to longer or full-length PGRN drug molecules. In some embodiments, off-target effects or other toxicities can be reduced by administering the GEM combinations of the present invention compared to full-length PGRN or to a combination of a greater number of GEMs. Therefore, in some embodiments, a GEM combination with 2, 3 or 4 GEMs can be superior to a more complex molecule with a combination of 5 or 6 GEMs. In some embodiments, a GEM combination with 2 or 3 GEMs can be superior to a more complex molecule with a combination of 4, 5 or 6 GEMs.

In an embodiment, a GEM combination may present two GEMs, such a combination may be referred to as a GEM dimer. A GEM combination may present three GEMs, such a combination may be referred to as a GEM trimer.

NSC34 Cell Proliferation:

In preferred embodiments, the GEMs and GEM combinations of the present invention show an enhancing effect on NSC34 cell proliferation. In embodiments, in addition to GEM F+E+D+C, these GEM combinations also involve GEM F+E, F+B, F+C and B+C, B+E and C+E. In embodiments, these GEM combinations involve F+E+B and F+E+C.

In an embodiment, a GEM combination comprising GEM E and one or more of GEM B, C, or E shows improved performance relative to full length PGRN or compared to other GEMs alone or in combination. In an embodiment, a GEM combination comprising GEM B and one or more of GEM C or E shows improved performance relative to full length PGRN or compared to other GEMs alone or in combination. In an embodiment, a GEM combination comprising GEM C and GEM E shows improved performance relative to full length PGRN or compared to other GEMs alone or in combination.

In embodiments, a GEM combination including a combination of GEM dimers or trimers of GEM E, C, B, and F appears to support cell proliferation of the NSC34 motor neuron-like cell line to a greater extent than full-length PGRN or other GEM treatments alone or in combination.

Cathepsin D maturation:

In preferred embodiments, the GEMs and GEM combinations of the invention exhibit improved performance relative to full-length PGRN in a cathepsin D maturation assay. In embodiments, these GEM combinations involve GEM F with one or more of E, D, or G, which show improved performance relative to full-length PGRN and compared to the other GEMs alone or in combination.

In an embodiment, a GEM combination comprising GEM B with one or more of C, E, G, A, or D shows improved performance relative to full-length PGRN or compared to other GEMs alone or in combination. In an embodiment, a GEM combination comprising GEM C with one or more of E, G, A, or D shows improved performance relative to full-length PGRN or compared to other GEMs alone or in combination. In an embodiment, a GEM combination comprising GEM E with one or more of G, A, or D shows improved performance relative to full-length PGRN or compared to other GEMs alone or in combination.

In an embodiment, a GEM combination comprising GEM G and one or more of A or D shows improved performance relative to full length PGRN or compared to other GEMs alone or in combination. In an embodiment, GEM A and/or D show improvements alone or in combination.

In an embodiment, a GEM combination comprising a double GEM combination of GEM B+D, C+D, E+D, G+D, A+D and GEM D shows enhanced cathepsin D action in motor neuron cells. In an embodiment, a GEM combination comprising a double GEM combination of GEM E, C, B and/or F shows enhanced cathepsin D action in motor neuron cells.

TDP-43 accumulation:

In preferred embodiments, the GEMs and GEM combinations of the invention show improved performance in reducing TDP-43 accumulation relative to full-length PGRN. In embodiments, a GEM combination comprising GEM F with one or more of E, D, A, or G shows improved performance relative to full-length PGRN or compared to other GEMs, alone or in combination. In embodiments, a GEM combination comprising GEM B with A shows improved performance relative to full-length PGRN and compared to other GEMs, alone or in combination.

In embodiments, GEM combinations including GEM F+E, F+A or F+D appear to show an enhancing effect on the ability of motor neuron cells to properly clear TDP-43, even with known TDP-43 mutations. In other embodiments, these GEM combinations encompass GEM F+E+A and/or GEM F+E+D.

Small PGRN

In preferred embodiments, the GEMs and GEM combinations of the invention exhibit improved performance following stable genomic introduction into target cells.

In an embodiment, the present invention relates to so-called "mini-PGRNs", also referred to as GEM combinations corresponding to the amino-terminal half (GEM GFB) or the carboxyl-terminal half (GEM CDE) of PGRN or full-length human PGRN (hPGRN).

In embodiments, GEMs and GEM combinations of the invention provide protection against serum deprivation stress challenge. In embodiments, GEMs E and F provide protection. In embodiments, the protective activity in CDE is at least partially affected by the E module and/or the protective activity of GFB is at least partially affected by the F module.

Features:

In embodiments, GEMs and GEM combinations of the invention provide beneficial effects on survival using a TDP-43 cytotoxic challenge.

In embodiments, the GEMs and GEM combinations of the present invention provide beneficial effects in maintaining neuronal morphology upon serum deprivation stress.

In embodiments, the GEMs and GEM combinations of the invention provide beneficial effects on the rate of neurite extension.

In embodiments of the present invention, the beneficial functional properties demonstrated by the examples provided herein are applicable to each of the GEM combinations and any possible combination of GEMs and are considered to be disclosed in conjunction with each of the GEM combinations and any possible combination of GEMs, falling within the scope of the present disclosure. The disclosure of the present invention is intended such that each of the embodiments disclosed herein can be combined with other embodiments, for example, the functional or structural features disclosed for any given GEM combination can be applied to other GEM combinations.

Any disclosure of a combination of GEMs expressed with a "+" sign between two or more GEMs may relate to the combined presence of the GEMs in a recombinant protein or a corresponding nucleic acid encoding said combination, and/or to the combined administration or preparation by spatial proximity (e.g. in a kit) of the GEMs or corresponding nucleic acid molecules linked by a "+" sign.

Each of the GEM combinations disclosed in the context of a functional feature, e.g., with respect to a beneficial potential therapeutic effect, is considered to be disclosed in combination with the functional feature or independently. Each of the functional features disclosed herein may be applied to any of the disclosed GEMs or GEM combinations, and may be further supported by one or more of the experimental methods disclosed herein.

Other implementations:

Any GEM or combination of GEMs disclosed herein is considered disclosed in combination with its corresponding signal sequence and/or linker/leader sequence, as disclosed in the sequence listing below.

As disclosed herein in embodiments of the invention, recombinant polypeptides, whether administered as recombinant proteins or via gene therapy, can be configured for efficient delivery and therapeutic efficacy.

In an embodiment, the recombinant polypeptide or combination of recombinant polypeptides does not include full-length PGRN.

In an embodiment, the recombinant polypeptide or combination of recombinant polypeptides does not comprise a truncation of the full-length PGRN.

In an embodiment, the recombinant polypeptide or combination of recombinant polypeptides includes a non-naturally occurring sequence, such as a recombinant or synthetic sequence, that is different from a naturally occurring PGRN sequence or a truncation or fragment thereof.

In an embodiment, the recombinant polypeptide or combination of recombinant polypeptides comprises a non-naturally occurring linker, leader and/or signal sequence, preferably a linker sequence that is different from the native order or a sequence present in the full-length naturally occurring PGRN.

In one embodiment, the recombinant polypeptide includes a signal sequence located at the N-terminus of the GEM.

In one embodiment, the recombinant polypeptide includes one or more linker sequences (also called leader sequences) located between the GEMs.

In these embodiments, the signal and/or linker/leader sequences can provide improved expression, folding, cleavage and/or activity of the GEM fragments, particularly when expressed in combination.

In one embodiment, the recombinant polypeptide comprises or consists of a mixed portion of the full-length PGRN sequence according to SEQ ID NO 1 and/or does not consist of the full-length PGRN sequence according to SEQ ID NO 1. These embodiments relate to a combination of GEMs selected from the full-length PGRN protein, but not to the exact set of full-length PGRN proteins. In an embodiment, the combination of GEMs described herein as a synthetic construct shows advantages over full-length PGRN therapy.

All embodiments presented herein involving a recombinant polypeptide or a combination of multiple recombinant polypeptides also relate to a therapeutic nucleic acid molecule or a combination of multiple therapeutic nucleic acid molecules. The GEM combinations disclosed herein can be administered, but are not limited to, as proteins, or via gene therapy, i.e., by administering nucleic acid molecules encoding one or more GEMs disclosed herein, or a combination thereof.

In embodiments, the present invention relates to recombinant polypeptides comprising 2-6 GEMs. In embodiments, the present invention relates to combinations of multiple recombinant polypeptides comprising 2-6 GEMs. Thus, the present invention relates to a single recombinant polypeptide or a combination of multiple recombinant polypeptides, any of which is defined by the presence of 2-6 GEMs. For example, 2-6 GEMs may be present in separate polypeptides, but may be present in combination, such as for combined administration to a subject. For example, 2-6 GEMs may be present in a single polypeptide.

These embodiments concerning 2-6 combined GEMs present in a single molecule or present in multiple molecules but combined also apply to the nucleic acid molecules of the invention.

In embodiments, the invention relates to a combination of multiple nucleic acid molecules encoding 2-6 GEMs. Thus, the invention relates to a single nucleic acid molecule encoding a GEM, or a combination of multiple nucleic acid molecules encoding a GEM, any of which is defined by the presence of two to six GEMs. For example, 2-6 GEMs may be present/encoded in separate nucleic acid molecules encoding the GEMs, but in combination, e.g., for administration in combination to a subject. For example, 2-6 GEMs may be present/encoded in a single nucleic acid molecule encoding the GEMs.

The nomenclature of GEMs used herein corresponds to the nomenclature established in the art.Alternative nomenclature may be provided herein.

Preferred sequences of the present invention relate to:

In an embodiment of the present invention, the recombinant polypeptide described herein is characterized in that:

a. GEM E comprises or consists of SEQ ID NO 5,

b. GEM F comprises or consists of SEQ ID NO 2,

c. GEM C comprises or consists of SEQ ID NO 4,

d. GEM D comprises or consists of SEQ ID NO 8,

e. GEM A comprises or consists of SEQ ID NO 7,

f. GEM B comprises SEQ ID NO 3 or consists of SEQ ID NO 3.

In an embodiment, the leader (signal) sequence comprises or consists of SEQ ID NO 26.

In an embodiment, the linker sequence comprises or consists of one or more of the sequences presented in the table above, which is located, for example, between the GEM coding regions, presented as unmarked sequences in constructs SEQ ID NOs 17-25, and found in SEQ ID NOs 27-35, as described above.

The present invention also relates to a recombinant polypeptide as described herein, which comprises or consists of: one or more of SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 36 and/or 37.

The present invention also relates to a recombinant polypeptide as described herein, which comprises or consists of one or more of SEQ ID NOs 27-35.

The present invention also relates to a nucleic acid molecule encoding a polypeptide as described herein, which comprises or consists of a sequence encoding one or more of SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 36 and/or 37.

The present invention also relates to a nucleic acid molecule as described herein, comprising or consisting of a sequence according to SEQ ID NO 17, 18, 19, 20, 21, 22, 23, 24 and/or 25.

The embodiments disclosed herein regarding GEM sequences further relate to sequence variants of the sequences, as disclosed in more detail below and in the detailed description. Each sequence is considered to include sequence variants having a percentage sequence identity of at least 70%, 75%, 80%, 85%, preferably 90%, more preferably at least 95% with the specific sequence. Each sequence is also considered to include sequence variants having length truncations or extensions, such as having 0 to 10 amino acid additions or deletions at either end of the sequence.

In one embodiment, the present invention encompasses the nucleic acid molecules described herein and various uses thereof, selected from the group consisting of:

a) a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding one or more of a combination of a plurality of granulin polypeptides selected from the group consisting of GEM A, GEM B, GEM C, GEM D, GEM E, GEM F and GEM G, or any other embodiment or combination of GEM disclosed herein, or a nucleic acid sequence of SEQ ID NOs 10-26,

b) a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding one or more of a combination of a plurality of granulin polypeptides selected from the group consisting of GEM A, GEM B, GEM C, GEM D, GEM E, GEM F and GEM G, wherein each GEM has a length between 20 and 100 amino acids, preferably between 40 and 70 amino acids,

c) a nucleic acid molecule complementary to a nucleotide sequence according to a) or b);

d) a nucleic acid molecule comprising a nucleotide sequence having sufficient sequence identity to a nucleotide sequence according to a), b) or c) to be functionally similar/equivalent, preferably comprising at least 70%, 80%, preferably 90%, more preferably 95% sequence identity to a nucleotide sequence according to a) or b);

e) a nucleic acid molecule which, due to the genetic code, is degenerate to a nucleotide sequence according to a) to d); and

f) A nucleic acid molecule of a nucleotide sequence according to a) to e), which is modified by deletion, addition, substitution, translocation, inversion and/or insertion and is functionally similar/equivalent to a nucleotide sequence according to a) to d).

Therefore, one embodiment of the present invention relates to a polypeptide as described herein or a combination thereof, comprising an amino acid sequence selected from the group consisting of:

a) the amino acid sequence of GEMA, GEM B, GEM C, GEM D, GEM E, GEM F and GEM G disclosed herein or any other embodiment or combination of GEM disclosed herein, or the amino acid sequence of SEQ ID NOs 2-8 or 27-35,

b) an amino acid sequence according to a) comprising 0 to 10 amino acid additions or deletions at the N- and/or C-terminus, c) an amino acid sequence comprising an amino acid sequence according to a) or b), wherein the length of the amino acid sequence is between 25 and 150 amino acids, preferably between 40 and 100 amino acids, most preferably between 50 and 70 amino acids,

c) an amino acid sequence having sufficient sequence identity to the amino acid sequence according to a) or b) to be functionally similar/equivalent, preferably comprising at least 70%, 80%, preferably 90%, more preferably 95% sequence identity to the amino acid sequence according to a); and

d) an amino acid molecule according to an amino acid sequence of a), b), c) or d), which is modified by deletion, addition, substitution, translocation, inversion and/or insertion and is functionally similar/equivalent to an amino acid sequence according to a), b), c) or d).

In another aspect, the present invention relates to a combination of recombinant polypeptides comprising two to six granulin/epithelial modules (GEMs).

In one embodiment, the combination of multiple recombinant polypeptides includes GEM E (4), and additionally one or more of GEM F (1), GEMC (3), and GEM D (7).

In another aspect, the present invention relates to a nucleic acid molecule encoding a recombinant polypeptide or a combination of multiple recombinant polypeptides as described herein.

In an embodiment, the nucleic acid molecule is present as a combination of multiple nucleic acid molecules, each nucleic acid molecule encoding one or more of the recombinant polypeptides as described herein.

In embodiments, a nucleic acid molecule as described herein is in the form of a vector configured to express a recombinant polypeptide upon administration to a subject.

In an embodiment, the vector is a viral vector. In an embodiment, the viral vector is selected from the group consisting of adenovirus, adeno-associated virus, lentivirus and baculovirus.

In embodiments of the invention, the combination of features including (a) a combination of GEMs less than full-length PGRN (including 2-6 GEMs) together with (b) a viral vector represents an improved and beneficial PGRN administration method compared to prior art methods such as administration of full-length PGRN. The reduced size of the GEM combination of the present invention, combined with a viral administration method, can potentially achieve improved delivery, transduction, expression, cutting and/or other functional improvements, thereby potentially achieving treatment or other functional improvements relative to full-length PGRN administration. Viral administration can also reduce toxicity compared to other modes of administration or compared to administration of full-length PGRN.

In an embodiment, the vector is an AAV vector, preferably an AAV9 vector, having an effective promoter, preferably a chicken B-actin modified promoter (CBh), and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

In embodiments, the nucleic acid molecule encodes a plurality of GEMs configured to be expressed as a polycistronic mRNA, wherein the GEMs are encoded by a single nucleic acid molecule and configured for post-transcriptional and/or post-translational cleavage, and/or wherein the polycistronic mRNA comprises multiple internal ribosome entry sites (IRES), enabling expression of a plurality of distinct and soluble GEM polypeptides.

In embodiments, the nucleic acid molecule encodes a plurality of GEMs configured for expression under the control of a plurality of promoters, enabling expression of a plurality of distinct and soluble GEM polypeptides.

Another aspect of the present invention relates to a pharmaceutical composition comprising a recombinant polypeptide, a combination of multiple recombinant polypeptides or a nucleic acid molecule as described herein and a pharmaceutically acceptable excipient.

In one embodiment, the present invention is present as a pharmaceutical combination, wherein

- (a.) the first GEM is in a pharmaceutical composition mixed with a pharmaceutically acceptable carrier, and (b.) the second and/or third GEM is in a separate pharmaceutical composition mixed with a pharmaceutically acceptable carrier, or

- (a.) the first GEM and (b.) the second and/or third GEM are present in the kit, in spatial proximity but in separate containers and/or compositions, or

- (a.) a first GEM and (b.) a second and/or a third GEM are combined in a single pharmaceutical composition admixed with a pharmaceutically acceptable carrier.

Another aspect of the present invention relates to a method of treating a neurodegenerative disease in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a recombinant polypeptide, a combination of multiple recombinant polypeptides, or a nucleic acid molecule described herein.

In an embodiment, the neurodegenerative disease to be treated is selected from the group consisting of: a motor neuron disease, such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), spinal muscular atrophy (SMA), Alzheimer's disease (AD) and Parkinson's disease (PD).

In an embodiment, the neurodegenerative disease to be treated is selected from the group consisting of: dementia, schizophrenia, epilepsy, stroke, poliomyelitis, neuritis, myopathy, hypoxia, anoxia, asphyxia, cardiac arrest, oxygen and nutrient deficiency in the brain after chronic fatigue syndrome, various types of intoxication, anesthesia, in particular neuroleptic anesthesia, spinal cord diseases, inflammation, in particular central inflammatory disorders, postoperative delirium and/or subsyndromal postoperative delirium, neuropathic pain, alcohol and drug abuse, alcohol and nicotine addiction, and/or the effects of radiation therapy.

In an embodiment, the neurodegenerative disease to be treated is selected from the group consisting of diseases associated with abnormal lysosomal function, such as Parkinson's disease (PD), Gaucher disease, or neuronal ceroid lipofuscinosis (NCL).

In an embodiment, the brain disorder to be treated is selected from schizophrenia and bipolar disorder.

In an embodiment, the disease to be treated is selected from peripheral inflammatory disorders, such as arthritis and atherosclerosis.

In an embodiment, the present invention relates to a therapeutically effective amount of a polypeptide or combination of the present invention, a combination of multiple recombinant polypeptides of the present invention, a nucleic acid molecule of the present invention, or a composition of the present invention for use as a medicament for treating a neurodegenerative disease.

In further aspects and embodiments, the present invention relates to:

- A GEM polypeptide selected from the group consisting of GEM A, GEM B, GEM C, GEM D, GEM E, GEM F and GEM G.

- A combination of a plurality of GEM polypeptides selected from the group consisting of GEM A, GEM B, GEM C, GEM D, GEM E, GEM F and GEM G.

- A combination of two GEM polypeptides selected from the group consisting of GEM A, GEM B, GEM C, GEM D, GEM E, GEM F and GEM G.

- A combination of three or more GEM polypeptides selected from the group consisting of GEM A, GEM B, GEM C, GEM D, GEM E, GEM F and GEM G.

In further aspects and embodiments, the present invention relates to:

- a nucleic acid molecule encoding a polypeptide selected from the group consisting of GEM A, GEM B, GEM C, GEM D, GEM E, GEM F and GEM G.

- A nucleic acid molecule encoding a combination of a plurality of GEM polypeptides selected from the group consisting of GEM A, GEM B, GEM C, GEM D, GEM E, GEM F and GEM G.

- A nucleic acid molecule encoding a combination of two GEM polypeptides selected from the group consisting of GEM A, GEM B, GEM C, GEM D, GEM E, GEM F and GEM G.

- A nucleic acid molecule encoding a combination of three or more GEM polypeptides selected from the group consisting of GEM A, GEM B, GEM C, GEM D, GEM E, GEM F and GEM G.

In further aspects and embodiments, the present invention relates to:

- A pharmaceutical combination comprising a plurality, preferably 2 or 3 GEM polypeptides, or comprising nucleic acid molecules encoding a plurality, preferably 2 or 3 GEM polypeptides.

- A polypeptide, a nucleic acid molecule, a pharmaceutical composition or a combination thereof as described herein, preferably using a GEM according to the sequence of SEQ ID NO 2, 3, 4, 5, 6, 7, 8, 36 and/or 37, and/or sequence variants thereof.

The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise form disclosed. Although the specific embodiments and examples of the present disclosure are described herein for the purpose of illustration, various equivalent modifications can be made within the scope of the present disclosure as will be appreciated by those skilled in the relevant art. For example, although the method steps or functions are presented in a given order, alternative embodiments can perform functions in different orders, or can perform functions substantially simultaneously. The teachings of the disclosure provided herein can be appropriately applied to other programs or methods. The various embodiments described herein can be combined to provide further embodiments. If necessary, various aspects of the present disclosure can be modified to provide another embodiment of the present disclosure using the composition, function and concept of the above-mentioned references and applications. In addition, for the consideration of biological functional equivalence, some changes can be made to the protein or nucleic acid sequence or structure without affecting the type or amount of biological or chemical action. These and other changes can be made to the present disclosure according to the detailed description. All these modifications are intended to be included within the scope of the appended claims.

The specific features of any of the foregoing embodiments may be combined or substituted for elements in other embodiments. In addition, although the advantages associated with certain embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments must exhibit such advantages to fall within the scope of the present disclosure.

DETAILED DESCRIPTION

The present invention relates to methods and compositions comprising granulin/epithelialin modules (GEMs) or combinations thereof suitable for treating neurodegenerative diseases. The present invention also relates to methods of treating neurodegenerative diseases, such as methods of administering therapeutic recombinant GEM proteins or gene therapy for delivering recombinant GEM gene products.

All patents and other publications cited in this application, including literature references, published patents, published patent applications, and co-pending patent applications, are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, methods described in such publications that can be used in conjunction with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application.

Progranulin and Granulin/Epithelialin Module (GEM):

Progranulin (PGRN) is a widely expressed secreted glycoprotein that serves as a trophic factor for a variety of cell types, including neurons. PGRN regulates inflammation and promotes wound repair. PGRN is involved in the regulation of a variety of processes, including development, wound healing, angiogenesis, growth and maintenance of neuronal cells, and inflammation. In microglia, progranulin is continuously expressed and secreted. Progranulin in neurons is important for the normal transport and function of lysosomal enzymes, such as β-glucocerebrosidase and cathepsin D.

Changes in PGRN expression have been found in a variety of neurodegenerative diseases, and studies on the genetic etiology of neurodegenerative diseases have shown that inherited mutations in the PGRN gene may lead to adult onset of neurodegenerative diseases due to reduced neuronal survival. Complete PGRN deficiency and loss-of-function mutations lead to neurodegenerative diseases or disorders, such as familial frontotemporal dementia (FTD) and neurodegenerative lysosomal storage diseases neuronal ceroid lipofuscinosis (NCL), including neuronal ceroid lipofuscinosis 11 (CLN11). Low PGRN promotes neuroinflammation and enhances peripheral inflammatory conditions, such as arthritis and atherosclerosis, and therefore any disease characterized by neuroinflammation or peripheral inflammation may be a disease that can be potentially treated using PGRN. In particular, in addition to AD and PD, low PGRN is also a risk factor for schizophrenia, bipolar disorder, and mental illness.

As used herein, the term "progranulin" or "PGRN" is primarily used, although in some embodiments "progranulin" or "PGRN" may be used as a synonym for the terms "proepithelin," "acrogranin," and "GP80," which may be used interchangeably herein.

Progranulin is the precursor protein of granulin (GEM). Cleavage of progranulin produces various active approximately 6 kDa granulin (GEM) peptides. These smaller cleavage products are named granulin A, granulin B, granulin C, etc., or GEMA, GEM B, GEM C, etc. Epithelin 1 and 2 are synonymous with granulin A and B, respectively.

As used herein, the terms "granulin", "granulin/epithelin module", "GEM", "epithelin" or "Grn" are used interchangeably and refer to the GEMs of the present invention. Reference to a "granulin polypeptide" also refers to a GEM polypeptide or a combination of GEM polypeptides, as described herein. For example, administration of a "granulin polypeptide" can be used in the described embodiments of administering a "combination of GEMs".

The cleavage of progranulin into granulin occurs in the extracellular matrix or lysosomes. Elastase, proteinase 3 and matrix metalloproteinases are proteases that can cleave progranulin into individual granulin peptides. Each individual granulin domain peptide is about 60 amino acids in length. Granulin peptides are rich in cysteine and can form 6 disulfide bonds/residues. The disulfide bonds form a central rod-shaped core that shuttles each individual granulin peptide into a stacked β-sheet configuration. In humans, seven GRN (1-7) modules (GEMs) are present in a precursor protein called progranulin (PGRN) in tandem repeats. Each GRN (GEM) domain consists of 12 cysteines located in conserved positions. A review of granulin/GEMs is provided in Tolkatchev et al, Protein Sci. 2008 Apr; 17 (4): 711-724.

The GEM nomenclature used in this disclosure and the Examples may include alternative numbering schemes, as outlined in the Examples.

The present invention encompasses various combinations of two or three or more GEM/granulin proteins. As examples, possible combinations are provided below. The positions in the combinations may be restrictive or non-restrictive, i.e., in some embodiments, the positions of the granulin proteins may be present in the recombinant construct in the order presented, although the order may vary.

The combination of GEMs can present two GEMs, and such a combination can be called a GEM dimer. The combination of GEMs can present three GEMs, and such a combination can be called a GEM trimer.

The combination of the two granulin proteins is as follows:

Potential combinations of the three granulin proteins are as follows:

Definition/Numerator:

As used herein, "nucleic acid" or "nucleic acid molecule" means a molecule composed of a chain of monomeric nucleotides, such as, for example, a DNA molecule (e.g., cDNA or genomic DNA). Nucleic acid can encode, for example, a promoter, a PGRN gene or a portion thereof, or a regulatory element. Nucleic acid molecules can be single-stranded or double-stranded.

"PGRN nucleic acid" or "GEM nucleic acid" refers to a nucleic acid comprising the following: a PGRN gene or its GEM portion, or a functional variant of a PGRN gene or its GEM portion. Functional variants of a gene include gene variants with minor changes (such as, for example, silent mutations, single nucleotide polymorphisms, missense mutations, and other mutations or deletions that do not significantly alter gene function).

As used herein, the term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified in a manner not found in nature to contain a nucleic acid fragment, or is synthetic. When the nucleic acid construct contains the control sequences required for expression of the coding sequence of the present disclosure, the term nucleic acid construct is synonymous with the term "expression cassette".

A DNA sequence that "encodes" a specific PGRN protein (including fragments and portions thereof) is a nucleic acid sequence that is transcribed into a specific RNA and/or protein. A DNA polynucleotide can encode an RNA (mRNA) that is translated into a protein, or a DNA polynucleotide can encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, or DNA targeting RNA; also referred to as "non-coding RNA" or "ncRNA").

As used herein, the term "gene" or "coding sequence" refers broadly to a region of DNA that encodes a protein (transcribed region). When a coding sequence is placed under the control of an appropriate regulatory region (such as a promoter), the coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide. A gene may include multiple operably linked segments, such as a promoter, a 5'-leader sequence, a coding sequence, and a 3'-untranslated sequence, including a polyadenylation site. The phrase "expression of a gene" refers to the process by which a gene is transcribed into RNA and/or translated into an active protein.

As used herein, "polypeptide" shall refer to both peptides and proteins. In the present invention, polypeptides may be naturally occurring or recombinant (i.e., produced via recombinant DNA technology), and may contain mutations (e.g., point mutations, insertion mutations, and deletion mutations) and other covalent modifications (e.g., glycosylation and labeling (via biotin, streptavidin, fluorescein, and radioisotopes)) or other molecular bonds with additional components. For example, pegylated proteins are encompassed within the scope of the present invention. Pegylation has been widely used as a post-production modification method for improving the biomedical efficacy and physicochemical properties of therapeutic proteins. The applicability and safety of this technology have been demonstrated by the use of various pegylated drugs over the years (see Jevsevar et al, Biotechnol J. 2010 Jan; 5 (1): 1 13-28). In some embodiments, the polypeptides described herein are modified to exhibit a longer in vivo half-life and resist degradation compared to unmodified polypeptides. Such modifications are known to those skilled in the art, such as cyclized polypeptides, polypeptides fused to vitamin B12, stapled peptides, protein lipidation, and replacement of natural L-amino acids with D-amino acids (see Bruno et al, Ther Deliv. 2013 Nov; 4(11): 1443-1467).

As used herein, a "secretion signal sequence" or "signal peptide" (sometimes referred to as a signal sequence, targeting signal, localization signal, localization sequence, transit peptide) is a short peptide (usually 16-30 amino acids long) that is present at the N-terminus (or occasionally the C-terminus) of most newly synthesized proteins destined to enter the secretory pathway. These proteins include those that reside in certain organelles (endoplasmic reticulum, Golgi apparatus, or endosomes), are secreted from cells, or are inserted into most cell membranes. Although most type I membrane-bound proteins have signal peptides, most type II and multi-spanning membrane-bound proteins are targeted to the secretory pathway through their first transmembrane domain, which is biochemically similar to a signal sequence, except that it is not cleaved. They are a kind of target peptide. The role of the signal peptide is to prompt the cell to transfer the protein generally to the cell membrane. In prokaryotes, the signal peptide guides the newly synthesized protein to the SecYEG protein conduction channel present in the plasma membrane. There is a homologous system in eukaryotes, in which the signal peptide guides the newly synthesized protein to the Sec6 channel, which has structural and sequence homology with SecYEG, but is present in the endoplasmic reticulum. SecYEG and Sec61 channels are often referred to as transporters, and transport through this channel is called translocation. When a secreted protein passes through the channel, the transmembrane domain may diffuse through the side gate in the transporter and be partitioned into the surrounding membrane.

In some embodiments, the expression construct is monocistronic (e.g., the expression construct encodes a single fusion protein including a first gene product and a second gene product). In some embodiments, the expression construct is polycistronic (e.g., the expression construct encodes two different gene products, such as two different proteins or protein fragments).

The polycistronic expression vector may include one or more (e.g., 1, 2, 3, 4, 5 or more) promoters. Any suitable promoter may be used, such as a constitutive promoter, an inducible promoter, an endogenous promoter, a tissue-specific promoter (e.g., a CNS-specific promoter), etc. In some embodiments, the promoter is a chicken β-actin promoter (CBA promoter), a CAG promoter (e.g., as described in Alexopoulou et al. (2008) BMC Cell Biol. 9:2; doi: 10.1186/1471-2121-9-2), a CD68 promoter, or a JeT promoter (e.g., as described in Tornoe et al. (2002) Gene 297 (1-2): 21-32). In some embodiments, the promoter is operably linked to a nucleic acid sequence encoding a first gene product, a second gene product, or a first gene product and a second gene product. In some embodiments, the expression cassette includes one or more additional regulatory sequences, including but not limited to transcription factor binding sequences, intron splice sites, poly(A) addition sites, enhancer sequences, repressor binding sites, or any combination of the foregoing.

In some embodiments, the nucleic acid sequence encoding the first gene product and the nucleic acid sequence encoding the second gene product are separated by a nucleic acid sequence encoding an internal ribosome entry site (IRES). Examples of IRES sites are described in, for example, Mokrejs et al. (2006) Nucleic Acids Res. 34 (Database issue): D125-30. In some embodiments, the nucleic acid sequence encoding the first gene product and the nucleic acid sequence encoding the second gene product are separated by a nucleic acid sequence encoding a self-cleaving peptide. Examples of self-cleaving peptides include, but are not limited to, T2A, P2A, E2A, F2A, BmCPV 2A and BmIFV 2A, and those described in Liu et al. (2017) Sci Rep. 7: 2193. In some embodiments, the self-cleaving peptide is a T2A peptide.

Sequence variation

The embodiments disclosed herein regarding GEM sequences further relate to sequence variants of the sequences, as disclosed in more detail below and in the detailed description. Each sequence is considered to include sequence variants having a percentage sequence identity of at least 70%, 75%, 80%, 85%, preferably 90%, more preferably at least 95% with the specific sequence. Each sequence is also considered to include sequence variants having length truncations or extensions, such as having 0 to 10 amino acid additions or deletions at either end of the sequence.

Protein modifications to the polypeptides of the present invention which may occur by substitution of the amino acid sequences and nucleic acid sequences encoding such molecules are also included within the scope of the present invention.

Replacement as defined herein is a modification of the amino acid sequence of a protein, wherein one or more amino acids are replaced by the same number of (different) amino acids, producing a protein comprising an amino acid sequence different from the original protein. In some embodiments, this modification does not significantly change the function of the protein. As with addition, replacement can be natural or artificial. As is well known in the art, amino acid replacement can be performed without significantly changing the function of the protein. This is especially true when the modification involves "conservative" amino acid replacement (i.e., replacing another amino acid with similar properties). Such "conservative" amino acids can be natural or synthetic amino acids, which can be replaced without significantly affecting the structure and function of the protein due to size, charge, polarity and conformation. Generally, many amino acids can be replaced by conservative amino acids without adversely affecting the function of the protein.

In general, the nonpolar amino acids Gly, Ala, Val, Ile and Leu, the nonpolar aromatic amino acids Phe, Trp and Tyr, the neutral polar amino acids Ser, Thr, Cys, Gln, Asn and Met, the positively charged amino acids Lys, Arg and His, and the negatively charged amino acids Asp and Glu represent conservative amino acid groups. This list is not comprehensive. For example, it is well known that Ala, Gly, Ser and sometimes Cys can be substituted for each other, even though they belong to different groups.

As is well known to those skilled in the art, any non-critical amino acid of a protein altered by conservative substitution should not significantly change the activity of the protein, because the side chain of the amino acid used to replace the natural amino acid should be able to form similar bonds and contacts with the side chain of the substituted amino acid. Non-conservative substitutions are possible as long as these substitutions do not excessively affect the neuroprotective or neurodegenerative activity of the polypeptide and/or reduce its effectiveness in treating neurodegenerative diseases.

As is well known in the art, "conservative substitutions" of amino acids or "conservative substitution variants" of polypeptides refer to amino acid substitutions that maintain the following: 1) the structure of the polypeptide backbone (e.g., beta sheet or alpha-helical structure); 2) the charge or hydrophobicity of the amino acid; and 3) the bulk of the side chain or any one or more of these characteristics. More specifically, the well-known term "hydrophilic residue" refers to serine or threonine. "Hydrophobic residue" refers to leucine, isoleucine, phenylalanine, valine or alanine. "Positively charged residue" refers to lysine, arginine or histidine. "Negatively charged residue" refers to aspartic acid or glutamic acid. Residues with "bulky side chains" refer to phenylalanine, tryptophan or tyrosine.

The term "conservative amino acid substitution" is well known in the art and involves replacing a particular amino acid with an amino acid having similar characteristics (e.g., similar charge or hydrophobicity, similar volume). Examples include replacing glutamic acid with aspartic acid, or replacing leucine with isoleucine. Conservative substitution variants will 1) have only conservative amino acid substitutions relative to the parent sequence, 2) will have at least 90% sequence identity, preferably at least 95% identity, 96% identity, 97% identity, 98% identity, or 99% or more identity relative to the parent sequence; and 3) will retain neuroprotective or neurorepairing activity. In this regard, any conservative substitution variant of the above-mentioned polypeptide sequences is contemplated according to the present invention. Such variants are considered to be "progranulin".

As used herein, "percent (%) sequence identity" relative to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to amino acid residues or nucleotides in a reference polypeptide or nucleic acid sequence, after aligning the sequences and introducing spaces (if necessary) to obtain the maximum percentage sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage amino acid or nucleic acid sequence identity can be achieved in various ways within the technical scope of the art, such as using publicly available computer software programs. Software such as BLAST or Clustal can achieve such sequence alignments and calculations of percentage identity. As used herein, the percentage homology between two sequences is equivalent to the percentage identity between the sequences. Percent identity or homology between sequences can be determined, for example, by using the GAP program (Genetics Computer Group, software; now available via Accelrys on http://www.accelrys.com), and alignment can be completed using, for example, the ClustalW algorithm (VNTI software, InforMax Inc.). Sequence databases can be searched using the target nucleic acid sequence. Database search algorithms are typically based on BLAST software (Altschul et al., 1990). In some embodiments, percent homology or identity can be determined along the entire length of the nucleic acid.

Nucleic acid molecules encoding GEMs of the present invention can be codon optimized according to standard methods in the art to express in cells comprising the target DNA of interest. For example, if the expected target nucleic acid is in human cells, it is contemplated that the codon-optimized polynucleotides encoding PGRN are used in constructs as described herein. According to some embodiments, the nucleotide sequence is codon optimized for mammalian expression.

The present invention also encompasses modified granulin sequences, i.e., sequences that differ from sequences encoding native granulin, as long as the modified sequence still encodes a protein that exhibits the biological activity of the native granulin at a higher or lower level of activity. These modified granulin sequences include modifications caused by point mutations, modifications due to genetic code degeneracy or naturally occurring allelic variants, and additional modifications introduced by genetic engineering to produce recombinant granulin nucleic acids.

In embodiments, the granulin nucleic acid comprises a nucleic acid having 95% homology to a sequence described herein, or a nucleic acid that hybridizes under highly stringent conditions to a complementary sequence of a DNA coding sequence of a granulin sequence described herein. As used herein, the term "hybridization" is used to refer to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (e.g., the strength of association between nucleic acids) are affected by factors such as the degree of complementarity between nucleic acids, the stringency of the conditions involved, the Tm (melting temperature) of the hybrid formed, and the G:C ratio within the nucleic acid. As used herein, the term "stringency" used refers to the temperature, ionic strength, and conditions of the presence of other compounds such as organic solvents under which nucleic acid hybridization is performed.

In an illustrative example, "high stringency conditions" can mean hybridization in 5X SSPE and 50% formamide at 65° C. and washing in 0.5X SSPE at 65° C. In another illustrative example, "high stringency conditions" can mean hybridization at 55° C. for 18 to 24 hours, and washing four times (5 min each) with 2x SSC, 1% SDS at room temperature, and then washing with 0.1x SSC at 50-55° C. for 15 min, the hybridization buffer consisting of: 50% formamide (vol/vol); 10% dextran sulfate; 1x Denhard solution; 20 mM sodium phosphate, pH 6.5; 5SSC; 200 μg salmon sperm DNA per ml hybridization buffer. In another illustrative example, conditions for high stringency hybridization are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", ^3rd Edition, Cold Spring Harbor Laboratory Press, (2001), which is incorporated herein by reference. In some illustrative aspects, hybridization occurs along the entire length of the nucleic acid. Detection of high stringency hybridization in the context of the present invention indicates strong structural similarity or structural homology (e.g., nucleotide structure, base composition, arrangement or order) with, for example, the nucleic acids provided herein.

As used herein, the term "complementarity" refers to the ability of purine and pyrimidine nucleotide sequences to associate to form double-stranded nucleic acid molecules by hydrogen bonds. Guanine and cytosine, adenine and thymine, and adenine and uracil are complementary, and when two nucleic acid molecules have "complementary" sequences, they can associate by hydrogen bonds to form double-stranded nucleic acid molecules. Complementary sequences can be DNA or RNA sequences. Complementary DNA or RNA sequences are referred to as "complementary sequences". Complementarity can be "partial" complementarity, in which only part of the nucleic acid bases match according to the base pairing rules, or, nucleic acids can be "completely" or "entirely" complementary.

In embodiments, with reference to the specific sequences provided herein, the polypeptide may have 0 to 10 amino acid additions or deletions at the N and/or C-terminus of the sequence, or the nucleic acid encodes a polypeptide that may have 0 to 10 amino acid additions or deletions at the N and/or C-terminus of the sequence. As used herein, the term "0 to 10 amino acid additions or deletions at the N and/or C-terminus of the sequence" means that the polypeptide may have a) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids at its N-terminus and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted at its C-terminus or b) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids at its C-terminus and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted at its N-terminus. or d) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted from its N-terminus and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted from its C-terminus.

In addition, in addition to the polypeptides described herein, peptide mimetics are also considered. Peptide analogs are often used in the pharmaceutical industry as non-peptide drugs, and their properties are similar to those of the template peptide. These types of non-peptide compounds are called "peptide mimetics" or "peptide mimics" (Fauchere (1986) Adv. Drug Res. 15: 29; Veber and Freidinger (1985) TINS p. 392; and Evans et al. (1987) J. Med. Chem. 30: 1229), and are usually developed with the help of computer molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce equivalent therapeutic or preventive effects. In some embodiments, it may be preferred to use peptide mimetics so as to prolong the stability of the polypeptide when administered to a subject. For this reason, peptide mimetics of polypeptides may be preferred.

Gene therapy and vectors used in gene therapy:

In various illustrative embodiments, the compositions described herein include (isolated and purified) nucleic acid sequences encoding granulin or a combination thereof. Methods for purifying nucleic acids are well known to those skilled in the art. In one embodiment, the sequence is operably linked to a regulatory sequence that directs the expression of granulin. In other embodiments, the sequence is operably linked to a heterologous promoter. In still other embodiments, the sequence is contained in a vector. In some embodiments, the vector is located in a host cell (e.g., a neuronal cell).

As used herein, the term "vector" is used to refer to a nucleic acid molecule that transfers one or more DNA or mRNA fragments to a patient's cells. A vector comprises a nucleic acid sequence and an appropriate nucleic acid sequence necessary for expressing an operably linked nucleic acid coding sequence in a patient. A vector is capable of expressing a nucleic acid molecule inserted into the vector and is capable of producing a polypeptide or protein. The nucleic acid sequence required for expression generally includes a promoter, an operator (optional), and a ribosome binding site, usually together with other sequences such as enhancers, terminators, and polyadenylation signals.

In another illustrative embodiment, the granulin nucleic acid can be introduced into a vector and administered to a patient by any protocol known in the art, such as those described in U.S. Pat. Nos. 6,333,194, 7,105,342, and 7,112,668, which are incorporated herein by reference. In an illustrative embodiment, the granulin nucleic acid can be introduced in vitro into cells extracted from a patient's organ, wherein the modified cells are then reintroduced into the body, or directly into appropriate tissues in vivo or using a targeted vector granulin nucleic acid construct. In various illustrative embodiments, the granulin nucleic acid can be introduced into cells or organs using, for example, viral vectors, retroviral vectors, or non-viral methods such as transfection, injection of naked DNA, electroporation, sonoporation, gene guns (e.g., by using high pressure gas to shoot DNA-coated gold particles into cells), synthetic oligomers, lipoplexes, polyplexes, virosomes, or dendrimers.

In one embodiment of treating cells or organs, a viral vector can be used to introduce granulin nucleic acid into cells or organs. The viral vector can be any viral vector known in the art. For example, the viral vector can be an adenoviral vector, a lentiviral vector, a retroviral vector, an adeno-associated viral vector, a herpes virus vector, a modified herpes virus vector, etc. In another illustrative embodiment of transfected cells, granulin nucleic acid can be introduced into cells by direct DNA transfection (liposome transfection, calcium phosphate transfection, DEAE-dextran, electroporation, etc.).

In various illustrative embodiments, the granulin nucleic acid can be, for example, a DNA molecule, an RNA molecule, a cDNA molecule, or an expression construct that includes a granulin nucleic acid.

The granulin nucleic acids described herein can be prepared or isolated by any conventional method commonly used to prepare or isolate nucleic acids, and include the nucleic acids of SEQ ID. No. (1) and (13). For example, DNA and RNA molecules can be chemically synthesized using commercially available reagents and synthesizers by methods known in the art. The granulin nucleic acids described herein can be purified by any conventional method commonly used in the art to purify nucleic acids. For example, electrophoresis methods and nucleic acid purification kits (e.g., Qiagen kits) known in the art can be used to purify granulin nucleic acids. Granulin nucleic acids suitable for delivery using viral vectors or introduction into cells by direct DNA transfection can also be prepared using any recombinant method known in the art.

The term "gene therapy" preferably refers to the transfer of DNA into a subject to treat a disease. Strategies for gene therapy using gene therapy vectors are known to those skilled in the art.

Such gene therapy vector is optimized to deliver exogenous DNA to the host cell of the subject. In a preferred embodiment, the gene therapy vector can be a viral vector. Viruses naturally develop strategies for integrating DNA into the host cell genome, and therefore can be used advantageously. Preferred viral gene therapy vectors may include but are not limited to retroviral vectors, such as Moloney murine leukemia virus (MMLV), adenovirus vectors, slow viruses, adenovirus-associated virus (AAV) vectors, poxvirus vectors, herpes simplex virus vectors or human immunodeficiency virus vectors (HIV-1). However, non-viral vectors may also be preferably used for gene therapy, such as plasmid DNA expression vectors driven by eukaryotic promoters or liposomes encapsulating transfer DNA. In addition, preferred gene therapy vectors may also refer to methods for transferring DNA, such as electroporation or direct injection of nucleic acid into a subject.

The isolated nucleic acid described herein can exist alone or as part of a vector. Generally, the vector can be a plasmid, a cosmid, a phasmid, a bacterial artificial chromosome (BAC) or a viral vector (e.g., an adenoviral vector, an adeno-associated virus (AAV) vector, a retroviral vector, a baculovirus vector, etc.). In some embodiments, the vector is a plasmid (e.g., a plasmid comprising an isolated nucleic acid as described herein). In some embodiments, the rAAV vector is single-stranded (e.g., single-stranded DNA). In some embodiments, the vector is a recombinant AAV (rAAV) vector. In some embodiments, the vector is a baculovirus vector (e.g., an Autographa californica nuclear polyhedron (AcNPV) vector).

The following paragraphs disclose various vectors. Bulcha, J.T., et al. (Viral vector platforms within the gene therapy landscape. Sig Transduct Target Ther 6, 53 (2021)) provides a useful review, the contents of which are incorporated in their entirety. The vectors disclosed therein are applicable to the present invention. Gene therapy is the treatment of genetic diseases by introducing genetic material that changes the function of specific cells into the patient's body. The key step in gene therapy is to effectively deliver genes to target tissues/cells, which is carried out by gene delivery vehicles called vectors. Modern viral vector-based gene therapy is achieved by delivering therapeutic genes in vivo to patients using vectors based on retroviruses, adenoviruses (Ads) or adeno-associated viruses (AAV).

Adenovirus (Ad) vector:

Ad is a non-enveloped virus known to cause infections of the upper respiratory tract primarily, but can also infect other organs, such as the brain and bladder. It possesses an icosahedral protein capsid that accommodates a 26 to 45 kb linear double-stranded DNA genome. The Ad genome is flanked by hairpin-shaped inverted terminal repeats (ITRs) of varying lengths (30-371 bp at its ends). The ITRs act as self-priming structures that promote primase-independent DNA replication. Packaging of the viral genome requires a packaging signal located on the left arm of the genome. The Ad genome encodes ~35 proteins that are expressed at early and late stages of viral gene transcription. The Ad genome includes five so-called "early" genes: E1A, E1B, E2, E3, and E4.7. The early genes are transcribed before viral DNA replication begins (approximately 7 h after infection). The "immediate early" E1A gene is essential for the transcription of other viral genes (e.g., E1B, E2, E3, and E4) that are responsible for viral DNA synthesis and play a role in regulating the expression of host genes. E1B plays a role in counteracting cellular activation of apoptosis by binding and inactivating p53, thereby allowing viral replication to proceed. The "late" genes (L1-L5) are generally required for viral assembly, release, and host cell lysis. These gene products are derived from five late transcription units generated by alternative splicing and polyadenylation of the major late messenger RNA.

Ad as a vector for gene therapy:

Ad vectors have the following advantages: (1) high transduction efficiency in both dormant and dividing cells; (2) persistence of epichromosomes in host cells; (3) broad tropism for different tissue targets; and (4) availability of scalable production systems. Modern Ad vectors are derived from human serotypes hAd2 and hAd5.

First generation. The first generation of Ad vectors were engineered by replacing the E1A/E1B region with a transgene cassette that could be up to 4.5 kb in length. Removal of the E1A gene renders the recombinant Ad (rAd) unable to replicate in host cells.

Second generation. Because of problems with the first generation of Ad vectors, researchers developed improved versions by further deleting other early gene regions (E2a, E2b, or E4), which provided additional space for larger transgene cassettes (10.5 kb). These new vector designs include temperature-sensitive rAd vectors, which were generated by eliminating the DNA binding protein encoded by E2A, deleting the DNA polymerase (Pol) protein encoded by E2b, and deleting the E4 region.

Third generation. Third generation Ad vectors, referred to as “gutless” or “helper-dependent” Ad vectors, have deleted all viral sequences except for the ITRs and packaging signals. These vectors, also referred to as “high-capacity” adenoviral vectors (HCAd), can accommodate ~36 kb of space for one or more cargo genes. Production of HAd in cell culture requires an additional adenoviral helper virus (HV), similar in composition to first generation vectors, but differing in that they contain loxP sites inserted flanking the packaging signal. Compared to previous generations of Ad vectors, HAd has reduced immunogenicity, prolonged transduction in host cells, and significantly larger cargo capacity, and can accommodate multiple transgene cassettes or therapeutic genes driven by their larger native promoters and enhancers to mimic physiological expression levels.

AAV vectors:

Adeno-associated virus (AAV) belongs to the Parvoviridae family and more specifically constitutes the Dependoparvovirus genus. As a dependoparvovirus, AAV lacks essential genes required for replication and expression of its own genome. These functions are provided by the Ad E1, E2a, E4, and VA RNA genes. The AAV genome itself is a single-stranded DNA containing four known open reading frames (ORFs). The first ORF encodes four replication genes (rep), named according to their molecular weight: Rep40, Rep52, Rep 68, and Rep78. The second ORF is the cap gene, encoding three viral capsid proteins, VP1, VP2, and VP3, respectively. The third and fourth are nested subgenomic mRNAs called assembly-activating proteins (AAPs), which are involved in the shuttling of capsid monomers to the nucleolus, where capsid assembly occurs; and the recently discovered membrane-associated accessory protein (MAAP), whose function is not fully understood. The 4.7 kb genome is flanked on both sides of the genome by 145 nt ITRs. The ITR serves as a self-priming structure for replication and provides the signal for Rep-mediated packaging.

AAV as a vector for gene therapy:

The first validation of an AAV vector for use in humans was performed in 1995 and involved the delivery of the cystic fibrosis transmembrane regulator (CFTR) gene (rAAV2-CFTR) packaged with an AAV2 capsid to patients with cystic fibrosis. Since the first validation, a variety of vector designs have been reported. The main consideration for AAV vector design is that the wild-type genome size is ∼4.7 kb. Therefore, vectors based on them are inevitably limited to a capacity of ∼5 kb. Therefore, all components required for correct expression need to be reduced/truncated/minimized to fit into the small capsid. Alternatively, strategies utilizing ITR-mediated recombination have produced dual-vector systems that can express "super-large" transgenes by means of transcriptional splicing of intermolecular recombinant ITRs from two complementary vector genomes. Other methods have also been developed to facilitate vector size expansion by protein "trans-splicing" via homology vector recombination, RNA trans-splicing 148 or via split design.

Vectors derived from AAV (i.e., recombinant AAV (rAVV) or AAV vectors) are attractive for delivering genetic material because (i) they are able to infect (transduce) a wide variety of non-dividing and dividing cell types, including myocytes and neurons; (ii) they lack viral structural genes, thereby attenuating host cell responses to viral infection, such as interferon-mediated responses; (iii) the wild-type virus is considered non-pathological in humans; (iv) in contrast to wild-type AAV, which is able to integrate into the host cell genome, replication-defective AAV vectors lack the rep gene and are typically present as episomes, thereby limiting the risk of insertional mutagenesis or genotoxicity; and (v) compared to other vector systems, AAV vectors are generally considered to be relatively poor immunogens and therefore do not elicit significant immune responses (see ii), thereby achieving persistence of vector DNA and potential long-term expression of therapeutic transgenes.

Typically, the rAAV vector (e.g., rAAV genome) includes a transgene (e.g., an expression construct, which includes one or more of the following: a promoter, an intron, an enhancer sequence, a protein coding sequence, an inhibitory RNA coding sequence, a poly A tail sequence, etc.) flanked by two AAV inverted terminal repeat (ITR) sequences. In some embodiments, the transgene of the rAAV vector includes an isolated nucleic acid as described in the present disclosure. In some embodiments, each of the two ITR sequences of the rAAV vector is a full-length ITR (e.g., a length of about 145bp and comprising a functional Rep binding site (RBS) and a terminal melting site (trs)). In some embodiments, one of the ITRs of the rAAV vector is truncated (e.g., shortened or non-full-length). In some embodiments, the truncated ITR lacks a functional terminal melting site (trs) and is used to produce a self-complementary AAV vector (scAAV vector). In some embodiments, the truncated ITR is an AITR, for example as described in McCarty et al. (2003) Gene Ther. 10(26):2112-8.

In some aspects, the present disclosure relates to a recombinant AAV (rAAV) comprising a transgenic nucleic acid encoding as described herein (e.g., an rAAV vector as described herein). The term "rAAV" generally refers to a viral particle comprising an rAAV vector encapsulated by one or more AAV capsid proteins. The rAAV described in the present disclosure may include a capsid protein having a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. In some embodiments, the rAAV described in the present disclosure includes a capsid protein as a wild-type capsid protein variant, such as a wild-type AAV capsid protein relative to its source, including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 (e.g., 15, 20, 25, 50, 100, etc.) amino acid substitutions (e.g., mutations). In some embodiments, the rAAV described in the present disclosure is easily spread through the CNS, particularly when introduced into the CSF space or directly into the brain parenchyma. Thus, in some embodiments, the rAAV described in the present disclosure comprises a capsid protein that is capable of crossing the blood-brain barrier (BBB).

In some embodiments, rAAV as described in the present disclosure (e.g., comprising a recombinant rAAV genome encapsidated by an AAV capsid protein to form rAAV capsid particles) is produced in a baculovirus vector expression system (BEVS). The use of BEVS to produce rAAV is described in, for example, Urabe et al. (2002) Hum Gene Ther 13 (16): 1935-43, Smith et al. (2009) Mol Ther 17 (11): 1888-1896, U.S. Pat. Nos. 8,945,918, 9,879,282, and International PCT Publication WO2017/184879. However, rAAV can be produced using any suitable method (e.g., using recombinant rep and cap genes).

Preclinical and clinical studies of rAAV gene delivery in the treatment of neurodegenerative, inherited, and acquired diseases affecting the nervous system are well established. The most studied in the CNS are serotypes 1, 2, 5, 8, 9, and recombinant human (rh) 10. The effectiveness of the serotype depends on the brain region, species, and target cell type. These serotypes effectively transduce neurons.

As disclosed in Hocquemiller et al, Human Gene Therapy, VOL 27 NUM 7 (2016), the following table describes various methods of rAAV therapy:

Any of the serotypes, promoters, dosage ranges, volumes or injection sites can be used in the present invention. For example, in embodiments of the present invention, NSE, CAG, PGK, CMV, Jet or CAG U1a promoters can be used. In embodiments, the injection site of rAAV can be selected from the white matter, subthalamic nucleus, striatum, putamen, substantia nigra, basal nucleus, intrathecal, lumbar or peripheral vein.

Lentiviral vectors:

Lentiviruses constitute a genus of the family Retroviridae. Retroviruses are spherical, enveloped, single-stranded RNA viruses with a diameter of ~100 nm. Lentivirus particles encapsidate two sense RNA strands bound by nucleocapsid proteins. The particles also contain reverse transcriptase, integrase, and protease proteins. Based on their genome structure, retroviruses can be divided into simple viruses and complex viruses. Gammaretroviruses are examples of simple retroviruses, while HIV-1 (a lentivirus) is an example of a complex retrovirus.

Lentivirus as a vector for gene therapy:

Lentiviral vectors have several features that make them suitable for transgene delivery for therapeutic purposes. Lentiviral vectors are integrating vectors that allow long-term transgene expression. They have a packaging capacity of up to 9 kb. High-level expression of multiple genes may be necessary to achieve therapeutic outcomes for certain diseases. Using two separate vectors carrying co-dependent transgenes may not be the best solution because the efficiency of successfully transducing multiple viral vectors into the same cell is not high. Lentiviral vectors have been shown to have the ability to express multiple genes from a single vector. Lentiviral vectors can transduce post-mitotic and dormant cells, while other retroviral-based platforms, such as gammaretroviral vectors, require active cell division for successful infection.

Lentiviral vector systems derived from HIV-1 virus have been developed for many years. These advances are partly intended to mitigate the potential risks associated with the virus on which the platform is based. The first generation of HIV-1-based vectors retained most of the viral genome in the trans-packaging construct, including the viral core, regulatory protein coding sequences, and auxiliary regulatory genes. Additional modifications were also made to improve the expression and transduction efficiency of lentiviral vectors. The introduction of transcriptional regulatory elements such as the central polypurine tract (cppt) and matrix attachment region (MAR) in cis expression vectors enhances viral transduction. In addition, the introduction of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) as a post-transcriptional regulatory element into the 3' untranslated region of the ORF significantly enhances transgene expression.

Composition, dosage and route of administration:

The polypeptides, nucleic acid molecules, gene therapy vectors or cells described herein may include different types of carriers depending on whether they are to be administered in solid, liquid or aerosol form, and whether they need to be sterile for routes of administration such as injection.

The active agents of the invention can be applied topically intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, topically, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, directly, via a catheter, via lavage, in an emulsion, in a lipid composition (e.g., liposomes), by sponge, or by other methods known to one of ordinary skill in the art, or any combination of the foregoing (see, e.g., Remingto's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

The present invention encompasses treating patients by introducing a therapeutically effective amount of polypeptides, nucleic acids, gene therapy vectors or cells into the bloodstream of a subject. As used herein, "introducing" a polypeptide, nucleic acid, gene therapy vector or cell into the bloodstream of a subject should include, but is not limited to, introducing such polypeptide, nucleic acid, gene therapy vector or cell into one of the veins or arteries of the subject via injection. Such administration may also be performed, for example, once, multiple times and/or over one or more extended periods. A single injection is preferred, but in some cases repeated injections may be required over time (e.g., quarterly, semi-annual or annual). Such administration is also preferably performed using a mixture of polypeptides, nucleic acids, gene therapy vectors or cells and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline.

As used herein, "pharmaceutically acceptable carriers" include any and all of the following: solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, their use in therapeutic compositions is contemplated. Supplementary active ingredients may also be introduced into the composition.

In addition, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions and emulsions, most preferably aqueous solutions. Aqueous carriers include water, alcohol/aqueous solutions, emulsions and suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution and non-volatile oils. Intravenous carriers include liquid and nutritional supplements, electrolyte supplements such as Ringer's dextrose, those based on Ringer's dextrose, etc. The liquid commonly used in i.v. administration, for example, can be found in Remington:The Science and Practice of Pharmacy, 2oth Ed., p.808, Lippincott Williams S-Wilkins (2000). Preservatives and other additives can also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, etc.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans. The preparation of aqueous compositions containing proteins as active ingredients is well known in the art. Generally, such compositions can be prepared as injectable liquid solutions or suspensions; they can also be prepared in a solid form suitable for dissolving or suspending in a liquid prior to injection. The formulation can also be emulsified.

Examples of parenteral dosage forms include aqueous solutions of the active agent in isotonic saline, 5% glucose or other well-known pharmaceutically acceptable liquid carriers (such as liquid alcohols, glycols, esters and amides). Parenteral dosage forms according to the present invention can be in the form of a reconstituted lyophilisate comprising a dose of a composition containing granulin. In one aspect of this embodiment, any of a variety of extended or sustained release dosage forms known in the art can be administered, such as, for example, a biodegradable carbohydrate matrix described in U.S. Pat. Nos. 4,713,249, 5,266,333 and 5,417,982, the disclosures of which are incorporated herein by reference.

In an illustrative embodiment, a general-purpose pharmaceutical formulation for parenteral administration is described with granulin, comprising: a) a pharmaceutically active amount of granulin; b) a pharmaceutically acceptable pH buffer to provide a pH in the range of about pH 4.5 to about pH 9; c) an ionic strength modifier in a concentration range of about 0 to about 250 millimolar; and d) a water-soluble viscosity modifier in a concentration range of about 0.5% to about 7% of the total formulation weight, or any combination of a), b), c), and d).

In various illustrative embodiments, pH buffers for use in the compositions and methods described herein are those known to those skilled in the art and include, for example, acetate, borate, carbonate, citrate, and phosphate buffers, as well as hydrochloric acid, sodium hydroxide, magnesium oxide, monopotassium phosphate, bicarbonate, ammonia, carbonic acid, hydrochloric acid, sodium citrate, citric acid, acetic acid, disodium hydrogen phosphate, borax, boric acid, sodium hydroxide, diethylbarbituric acid, and proteins, as well as various biological buffers, such as TAPS, Bicine, Tris, Tricine, HEPES, TES, MOPS, PIPES, Cacodylate, MES.

In another illustrative embodiment, ionic strength adjusting agents include those known in the art, such as glycerol, propylene glycol, mannitol, glucose, dextrose, sorbitol, sodium chloride, potassium chloride, and other electrolytes.

Useful viscosity modifiers include, but are not limited to, ionic and nonionic water-soluble polymers; cross-linked acrylic acid polymers, such as the "carbomer" family of polymers, which can be used as Carboxypolyalkylene commercially available under the trademark of TETRA; hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymers and polyvinyl alcohol; cellulose polymers and cellulose polymer derivatives such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, methylcellulose, carboxymethyl cellulose and etherified cellulose, etc.; gums such as tragacanth and xanthan gum; sodium alginate; gelatin, hyaluronic acid and its salts, chitosan, gellan gum or any combination thereof. Non-acidic viscosity enhancers such as neutral or alkaline agents are preferably used to facilitate achieving the desired pH of the formulation. If a uniform gel is required, a dispersant such as alcohol, sorbitol or glycerol may be added, or the gelling agent may be dispersed by grinding, mechanical mixing or stirring or a combination thereof. In one embodiment, the viscosity enhancer may also provide a base as described above. In a preferred embodiment, the viscosity modifier is a cellulose that has been modified such as etherified or esterified.

In various illustrative embodiments, provided granulin compositions may include compositions containing all or part of a granulin polypeptide, alone or in combination with at least one other agent (such as an excipient and/or a stabilizing compound and/or a solubilizing agent), and may be administered in any sterile, biocompatible drug carrier, including, but not limited to, saline, buffered saline, dextrose, glucose, and water. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starches from corn, wheat, rice, potatoes, etc.; celluloses, such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; and gums, including tragacanth; and proteins, such as gelatin and collagen. Suitable disintegrants or solubilizing agents include agar, alginic acid, or a salt thereof, such as sodium alginate.

In illustrative embodiments, the granulin polypeptide can be administered to a patient alone or in combination with other agents, drugs or hormones or in the form of a pharmaceutical composition mixed with one or more excipients or other pharmaceutically acceptable carriers. In one embodiment, the pharmaceutically acceptable carrier is pharmaceutically inert. In another embodiment, the granulin polypeptide can be administered alone to a patient suffering from a neurological disease.

The unit daily dosage of the composition comprising the granulin polypeptide can vary significantly depending on the patient's condition, the disease state being treated, the route of administration and tissue distribution of the granulin, and the possibility of co-use of other therapeutic treatments. The effective amount of granulin to be administered to the patient is based on body surface area, patient weight, the physician's assessment of the patient's condition, etc.

In an illustrative embodiment, an effective dosage of granulin can be in the range of about 1 ng/kg patient body weight to about 1 mg/kg patient body weight, more preferably about 1 ng/kg patient body weight to about 500 ng/kg patient body weight, and most preferably about 1 ng/kg patient body weight to about 100 ng/kg patient body weight.

In another illustrative embodiment, the effective dose of granulin polypeptide can be in the range of about 1 pg/kg patient weight to about 1 mg/kg patient weight. In various illustrative embodiments, the effective dose can be in the range of about 1 pg/kg patient weight to about 500 ng/kg patient weight, about 500 pg/kg patient weight to about 500 ng/kg patient weight, about 1 ng/kg patient weight to about 500 ng/kg patient weight, about 100 ng/kg patient weight to about 500 ng/kg patient weight, and about 1 ng/kg patient weight to about 100 ng/kg patient weight.

In another illustrative embodiment, the effective dose of granulin polypeptide can be in the range of about 1 μg/kg patient weight to about 1 mg/kg patient weight. In various illustrative embodiments, the effective dose can be in the range of about 1 μg/kg patient weight to about 500 μg/kg patient weight, about 500 ng/kg patient weight to about 500 μg/kg patient weight, about 1 μg/kg patient weight to about 500 μg/kg patient weight, about 0.1 μg/kg patient weight to about 5 μg/kg patient weight, about 0.1 μg/kg patient weight to about 10 μg/kg patient weight, and about 0.1 μg/kg patient weight to about 100 μg/kg patient weight.

In another illustrative embodiment, the effective dose of nucleic acid molecules can be in the range of about 1 million nucleic acid molecules per 70 kg patient body to about 1 billion nucleic acid molecules per 70 kg patient body. In various illustrative embodiments, the effective dose can be in the range of about 1 million nucleic acid molecules per 70 kg patient body to about 500 million nucleic acid molecules per 70 kg patient body, about 200,000 nucleic acid molecules per 70 kg patient body to about 200 million nucleic acid molecules per 70 kg patient body, about 1 million nucleic acid molecules per 70 kg patient body to about 200 million nucleic acid molecules per 70 kg patient body.

The composition comprising the granulin polypeptide may be suitable for parenteral administration, and the route of parenteral administration may be selected from the group consisting of intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, intraventricular, intrathecal, intracerebral and intracardiac.

In some embodiments, compositions are directly applied to the nervous system, CNS or peripheral nervous system of the experimenter, for example, by direct injection into the brain and/or spinal cord of the experimenter. The example of CNS direct administration mode includes but is not limited to intracerebral injection, intraventricular injection, intracisternal injection, intraparenchymal injection, intrathecal injection and any combination of the foregoing. In some embodiments, direct injection into the CNS of the experimenter causes transgenic expression (for example, the first gene product, the second gene product and if applicable, the expression of the third gene product) in the midbrain, striatum and/or cerebral cortex of the experimenter. In some embodiments, direct injection into the CNS causes transgenic expression (for example, the first gene product, the second gene product and if applicable, the expression of the third gene product) in the spinal cord and/or CSF of the experimenter.

As used herein, the term "nervous system" includes both the central nervous system and the peripheral nervous system. The term "central nervous system" or "CNS" includes all cells and tissues of the brain and spinal cord of vertebrates. The term "peripheral nervous system" refers to all cells and tissues of the nervous system part outside the brain and spinal cord. Therefore, the term "nervous system" includes but is not limited to cells in neuronal cells, glial cells, astrocytes, cerebrospinal fluid (CSF), cells in the interstitial space, cells in the spinal cord protective layer, epidural cells (i.e., cells outside the dura mater), cells in the non-neural tissues adjacent to or in contact with neural tissue or dominated by neural tissue, perineurium, perineurium, endoneurium, cord, bundle, etc. In some embodiments, polypeptide or nucleic acid is delivered to target cells, wherein the target cells preferably include neuronal cells.

In a preferred embodiment, the pharmaceutical composition used as a medicament as described herein is administered by introducing a therapeutically effective amount of the composition into the bloodstream of a subject. This route of administration is particularly advantageous for the administration of polypeptides. Advantageously, the polypeptides described herein, and particularly soluble polypeptides, can cross the blood-brain barrier. Therefore, systemic administration by introducing a therapeutically effective amount of a polypeptide into the vasculature can be used to treat neurodegeneration in the brain.

In a further preferred embodiment, the pharmaceutical composition described herein for use as a medicament is administered topically. It may also be preferred that topical administration of the pharmaceutical composition to the skin is achieved via a cream or lotion comprising the gene therapy or polypeptide.

In addition, in a preferred embodiment, the local application of polypeptide can be preferably mediated by implants such as collagen sponges. In a further preferred embodiment, polypeptide can be locally applied by hydrogel. Hydrogel is a three-dimensional cross-linked network of water-soluble polymers. Those skilled in the art know how to produce suitable hydrogels for delivering proteins or polypeptides (Hoare et al. 2008, Peppas et al. 2000, Hoffmann A. et al. 2012). In particular, the density of the cross-linked network of the hydrogel can be advantageously optimized to achieve a porosity suitable for loading the polypeptide into the hydrogel. Subsequently, the release of the polypeptide is controlled by the diffusion of the peptide in the entire gel network. Therefore, the release rate and therapeutically effective amount of the polypeptide can be accurately adjusted by optimizing the cross-linking density of the hydrogel. In addition, the preferred hydrogel can also encompass an outer membrane optimized for the release of the polypeptide. The preferred hydrogel is biocompatible and is preferably implanted to supply polypeptide locally for a long time.

Any effective regimen for administering a composition comprising granulin can be used. For example, a composition comprising granulin can be administered as a single dose, or a composition comprising granulin can be separated and administered as a multiple dose daily regimen. In addition, staggered regimens, such as one to three days per week, can be used as an alternative to daily treatment, and for purposes of the present invention, this intermittent or staggered daily regimen is considered to be equivalent to daily treatment and within the scope of the present invention. In one embodiment, a composition comprising granulin is used to treat a patient to reduce neuronal cell death with multiple injections. In another embodiment, a composition comprising granulin is injected to a patient multiple times (e.g., about 2 times to about 50 times) at intervals of, for example, 12-72 hours or at intervals of 48-72 hours. Additional injections of a composition comprising granulin can be administered to a patient at intervals of several days or months after one or more initial injections, and additional injections prevent the recurrence of the disease. Alternatively, one or more initial injections of a composition comprising granulin can prevent the recurrence of the disease.

In some embodiments, the composition is administered peripherally to the subject, such as by peripheral injection. Examples of peripheral injections include subcutaneous injection, intravenous injection, intraarterial injection, intraperitoneal injection, or any combination of the foregoing. In some embodiments, the peripheral injection is intraarterial injection, such as injection into the subject's carotid artery.

In some embodiments, the composition described in the present disclosure (e.g., a composition comprising an isolated nucleic acid or vector or rAAV) is administered to the CNS periphery or directly to the subject, or to the CNS periphery and directly to the subject. For example, in some embodiments, the composition is administered to the subject by intra-arterial injection (e.g., injection into the carotid artery) and/or by intraparenchymal injection (e.g., intraparenchymal injection by CED). In some embodiments, direct injection into the CNS and peripheral injection are simultaneous (e.g., occurring simultaneously). In some embodiments, direct injection occurs before peripheral injection (e.g., between 1 minute and 1 week before, or longer). In some embodiments, direct injection occurs after peripheral injection (e.g., between 1 minute and 1 week after, or longer).

The amount of the composition as described in the present disclosure (e.g., a composition comprising an isolated nucleic acid or vector or rAAV) administered to a subject will vary depending on the method of administration. For example, in some embodiments, rAAV as described herein is administered to a subject at a titer of between about 10 ⁹ genome copies (GC)/kg to about 10 ¹⁴ GC/kg (e.g., about 10 ⁹ GC/kg, about 10 ¹⁰ GC/kg, about 10 ¹¹ GC/kg, about 10 ¹¹ GC/kg, about 10 ¹² GC/kg, or about 10 ¹⁴ GC/kg). In some embodiments, a high titer (e.g., rAAV with >10 ¹² genome copies GC/kg) is administered to a subject by injection into the CSF space or by intraparenchymal injection.

The compositions described herein (e.g., compositions comprising isolated nucleic acids or vectors or rAAVs) can be administered to a subject one or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or more). In some embodiments, the composition is administered to a subject continuously (e.g., chronically), for example, via an infusion pump.

Medical Conditions:

In an embodiment, the neurodegenerative disease to be treated is selected from the group consisting of: motor neuron disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), spinal muscular atrophy (SMA), Alzheimer's disease (AD) and Parkinson's disease (PD). In other embodiments, the neurodegenerative disease to be treated is selected from the group consisting of: dementia, schizophrenia, epilepsy, stroke, poliomyelitis, neuritis, myopathy, hypoxia, anoxia, asphyxia, cardiac arrest, oxygen and nutrient deficiency in the brain after chronic fatigue syndrome, various types of poisoning, anesthesia, especially neuroleptic anesthesia, spinal cord disorders, inflammation, especially central inflammatory disorders, postoperative delirium and/or subsyndromal postoperative delirium, neuropathic pain, alcohol and drug abuse, alcohol and nicotine addiction, and/or the effects of radiation therapy. In an embodiment, the neurodegenerative disease to be treated is selected from the group consisting of: diseases associated with abnormal lysosomal function such as Parkinson's disease (PD), Gaucher disease or neuronal ceroid lipofuscinosis (NCL). In an embodiment, the brain disease to be treated is selected from schizophrenia and bipolar disorder. In an embodiment, the disease to be treated is selected from peripheral inflammatory disorders, such as arthritis and atherosclerosis.

In another illustrative embodiment, a pharmaceutical composition comprising a therapeutically effective amount of a granulin polypeptide or a combination thereof and a pharmaceutically acceptable carrier is provided, wherein the therapeutically effective amount comprises an amount capable of increasing neuronal cell survival and/or reducing neuronal cell death in a patient and/or reducing or preventing symptoms of a neurodegenerative disease in a patient.

In another illustrative embodiment, a method for reducing neuronal cell death in a patient is provided, the method comprising the step of administering to the patient a therapeutically effective amount of a granulin polypeptide or a combination thereof, wherein the amount of the peptide is effective to increase neuronal cell survival in the patient and/or reduce neuronal cell death in the patient and/or reduce or prevent symptoms of a neurodegenerative disease in the patient.

In another illustrative embodiment, a method for treating a patient suffering from a neurodegenerative disease is provided, the method comprising the steps of administering to the patient a composition comprising a granulin polypeptide or a combination thereof, and reducing neuronal cell death and/or reducing or preventing symptoms of the neurodegenerative disease in the patient.

In another illustrative embodiment, a pharmaceutical composition is provided comprising a therapeutically effective amount of a nucleic acid molecule expressing one or more granulin proteins and a pharmaceutically acceptable carrier, wherein the therapeutically effective amount comprises an amount capable of increasing neuronal cell survival and/or reducing neuronal cell death in a patient and/or reducing or preventing symptoms of a neurodegenerative disease in a patient.

In another illustrative embodiment, a method for reducing neuronal cell death in a patient is provided, the method comprising the step of administering to the patient a therapeutically effective amount of a nucleic acid molecule expressing one or more granulin proteins, wherein the amount of the nucleic acid molecule is effective to increase neuronal cell survival and/or reduce neuronal cell death in the patient and/or reduce or prevent symptoms of a neurodegenerative disease in the patient.

In another illustrative embodiment, a method for treating a patient suffering from a neurodegenerative disease is provided, the method comprising the steps of administering to the patient a composition comprising a nucleic acid molecule expressing one or more granulin proteins, and increasing neuronal cell survival and/or reducing neuronal cell death in the patient and/or reducing or preventing symptoms of the neurodegenerative disease in the patient.

In one illustrative aspect, the neurodegenerative disease state can include, but is not limited to, Parkinson's disease and parkinsonian syndromes, including progressive supranuclear palsy, Alzheimer's disease, and motor neuron disease (e.g., amyotrophic lateral sclerosis); or any other neurodegenerative disease mediated by increased neuronal cell death and/or altered progranulin expression and/or function.

In illustrative embodiments, neurodegenerative diseases are mediated by heritable mutations of the progranulin gene that modifies progranulin expression. For example, frontotemporal dementia (FTD), or frontotemporal degenerative diseases, or frontotemporal neurocognitive disorders cover several types of dementia involving the frontal and temporal lobes. FTD is widely manifested as behavioral or language disorders. Three major subtypes or variant syndromes are behavioral variants (bvFTD), formerly known as Pick's disease, and two variants of primary progressive aphasia-semantic variants (svPPA) and non-fluent variants (nfvPPA). Two rare different subtypes of FTD are neuronal intermediate filament inclusion disease (NIFID) and basophilic inclusion disease. Other related diseases include cortical basal syndrome and FTD with amyotrophic lateral sclerosis (ALS) FTD-ALS, also known as FTD-MND.

In 2006, progranulin was discovered as the major genetic cause of frontotemporal dementia (FTD), and only a few months earlier, TDP-43 was identified as the major protein component of histopathological lesions in the same patients. Until now, the only known FTD gene was MAPT, located near the progranulin gene GRN on chromosome 17q21, and these two discoveries broke a double impasse in the field. Approximately 70 GRN mutations are known to explain all 17q21-linked autosomal dominant FTD families that are not explained by tau mutations, and since all FTD patients with GRN mutations have TDP-43 pathology, TDP-43 explains the tau-negative protein inclusions in these families. GRN mutations explain up to 20% of familial FTD and 5% of sporadic FTD. Despite the histopathological commonalities, GRN mutations lead to a wide variety of clinical presentations, primarily causing behavioral FTD and progressive nonfluent aphasia, but also rarely with manifestations of Alzheimer's disease or Parkinsonism.

All pathological GRN mutations reduce progranulin levels or cause loss of function. In fact, blood progranulin levels indicate the presence of pathogenic progranulin mutations and are rapidly becoming a diagnostic biomarker. Progranulin is a secreted growth factor known for its role in biological processes such as inflammation, wound healing and cancer and its neurotrophic properties. It is proteolytically processed into peptides called granulins. Therefore, in some embodiments, the present invention is based on improved granulin treatment for FTD.

Several factors are known to influence the expression of progranulin. They include intrinsic factors, such as the gene TMEM106B and various microRNAs, as well as pharmaceutical agents, such as the histone deacetylase inhibitor SAHA and certain alkalinizing drugs. Agents that target the endocytic progranulin receptor sortilin-1 appear to increase plasma progranulin levels by slowing its internalization. Homozygous GRN mutations cause the rare lysosomal storage disease ceroid lipofuscinosis, and progranulin is localized to neuronal endomembrane compartments, including lysosomes. Both homozygous and heterozygous GRN knockout mice exist; the former exhibit behavioral and inflammatory phenotypes, and the latter exhibit only the former.

Frontotemporal dementias are mostly early-onset syndromes associated with frontotemporal lobar degeneration (FTLD) and are characterized by progressive neuronal loss that primarily involves the frontal or temporal lobes and typically involves more than 70% loss of fusiform neurons, while other neuronal types remain intact. FTD was first described by Arnold Pick in 1892 and was originally called Pick's disease, a term now used only to refer to the behavioral variant of FTD, which exhibits the presence of Pick bodies and cells. FTD is second only to Alzheimer's disease (AD) in prevalence, accounting for 20% of early-onset dementia cases. Signs and symptoms typically appear in late adulthood, more commonly between the ages of 45 and 65, and affect men and women roughly equally. Common signs and symptoms include marked changes in social and personal behavior, apathy, blunted emotions, and deficits in both expressive and receptive language. Currently, there is no cure for FTD, but there are treatments that can help relieve symptoms.

In illustrative embodiments, the neurodegenerative disease is mediated by environmental damage to the patient. As used herein, a neurodegenerative disease mediated by environmental damage to the patient means a disease caused by environmental damage and is not caused by a heritable mutation of the progranulin gene that modifies progranulin expression. A heritable mutation is a permanent mutation in the patient's DN that may be passed on to the patient's offspring.

However, these illustrative embodiments are not meant to exclude the influence of allelic variants of modifier genes, which are, for example, involved in the metabolism of neurotoxins, which make individuals more or less susceptible to the development of neurodegenerative diseases. As used herein, these modifier genes can change the course of disease development.

Neurodegenerative diseases mediated by environmental damage to the patient may be sporadic diseases related to environmental factors, which directly or indirectly cause neuronal cell death by changing gene expression. In various other illustrative embodiments, environmental damage is derived from the patient's diet or the result of endogenous synthesis, or both. In an illustrative embodiment, environmental damage causes synthesis of compounds that cause harmful effects in vivo. Neuronal cell death can occur by any multiple modes including but not limited to excitotoxicity or oxidative stress.

In another illustrative embodiment, the neurodegenerative disease state is mediated by excitotoxins. Excitotoxins are a class of substances that damage neurons by causing neuronal cell death through excessive activation of receptors (e.g., excitatory neurotransmitter glutamate receptors). Examples of excitotoxins include excitatory amino acids, which can produce lesions in the central nervous system. Other examples of excitotoxins include, but are not limited to, sterol glucosides, including β-sitosterol-β-D-glucoside and cholesterol glucoside, methionine sulfenyl imide, and other substances known in the art that induce neurological excitotoxic reactions in patients. In an illustrative embodiment, the excitotoxin is a sterol glycoside. In other illustrative embodiments, the sterol glycoside is selected from the group consisting of β-sitosterol-β-D-glucoside and cholesterol glucoside, or its analogs or derivatives.

In embodiments, the present invention can be used to treat nervous system diseases. As used herein, the term "nervous system disease" or disorder refers to any disorder of the nervous system. Structural, biochemical or electrical abnormalities of the brain, spinal cord or other nerves can lead to a range of symptoms. Examples of symptoms include paralysis, muscle weakness, poor coordination, loss of sensation, convulsions, confusion, pain, limited cognitive ability and altered level of consciousness. There are many recognized nervous system disorders, some of which are relatively common, but many are rare. They can be evaluated by neurological examinations and studied and treated within the specialties of neurology and clinical neuropsychology.

Alzheimer's disease (AD), also known simply as Alzheimer's, is a chronic neurodegenerative disease that gradually worsens over time. It is the cause of 60-70% of dementia cases. The most common early symptom is difficulty remembering recent events. As the disease progresses, symptoms may include language problems, disorientation (including getting lost easily), mood swings, loss of motivation, inability to care for oneself, and behavioral problems.

Parkinson's disease (PD), or simply Parkinson's, is a long-term degenerative disorder of the central nervous system that primarily affects the basal ganglia nerves that control movement. As the disease worsens, non-motor symptoms become more common. Early in the disease, the most obvious symptoms are tremors, rigidity, slow movements, and difficulty walking. Problems with thinking and behavior may also occur. Dementia becomes common in the late stages of the disease. The main motor symptoms are collectively referred to as "parkinsonism" or "parkinsonism."

An example of motor neuron disease is amyotrophic lateral sclerosis (ALS). ALS mainly involves the loss of spinal cord and cortical motor neurons, resulting in increased paralysis and ultimately death. Early symptoms of ALS include, but are not limited to, foot drop or weakness in the patient's legs, feet or ankles, weakness or clumsiness in the hands, muscle cramps, and twitching of the arms, shoulders, and tongue. ALS typically affects chewing, swallowing, speaking, and breathing, and eventually leads to paralysis of the muscles required to perform these functions. Shaw et al., Neuroscience and Biobehavioral Reviews, 27:493 (2003) describes a review of various nervous system diseases, which is incorporated herein by reference. The methods and compositions of the present invention can be used for human clinical medicine and veterinary applications. The methods and compositions described herein can be used alone or in combination with other methods or compositions.

Spinal muscular atrophy (SMA) is a rare neuromuscular disorder that causes loss of motor neurons and progressive muscle wasting. It is usually diagnosed in infancy and, if left untreated, is a common genetic cause of death in children. It may also appear later in life and then be less severe. Its common feature is the gradual weakness of voluntary muscles, with the arms, legs and respiratory muscles being affected first. Related problems may include poor head control, difficulty swallowing, scoliosis and joint contractures. Spinal muscular atrophy is caused by abnormalities (mutations) in the SMN1 gene that encodes SMN, a protein that is essential for the survival of motor neurons. The loss of these neurons in the spinal cord hinders signaling between the brain and skeletal muscles. Another gene, SMN2, is considered a disease-modifying gene because, generally, the more copies of SMN2 there are, the milder the course of the disease. The diagnosis of SMA is based on symptoms and confirmed by genetic testing.

In embodiments, the present invention can be used to treat lysosomal storage diseases. Lysosomal storage diseases (LSDs) are a group of inherited metabolic disorders, most of which are caused by enzyme deficiencies within lysosomes that lead to accumulation of undegraded substrates. This storage process can lead to a wide range of clinical manifestations, depending on the specific substrate and site of accumulation. Examples of LSDs include mucopolysaccharidosis, mucolipidosis, oligosaccharidosis, glycogen storage disease type II, Gaucher disease, Fabry disease, Niemann-Pick disease, and neuronal ceroid lipofuscinosis.

Gaucher disease is a rare inborn error of glycosphingolipid metabolism caused by a deficiency of the lysosomal enzyme acid β-glucocerebrosidase (Gcase, "GBA"). Patients suffer from non-CNS symptoms and manifestations, including hepatosplenomegaly, bone marrow insufficiency leading to pancytopenia, pulmonary disease and fibrosis, and bone defects. In addition, a significant number of patients suffer from neurological symptoms, including saccadic eye movement and gaze defects, seizures, cognitive deficits, developmental delays, and movement disorders (including Parkinson's disease). In addition to patients with Gaucher disease (mutations in both chromosomal alleles of the GBA1 gene), patients with mutations in only one allele of GBA1 are at a greatly increased risk of developing Parkinson's disease (PD). The severity of PD symptoms (including gait difficulties, resting tremor, rigidity, and often depression, sleep difficulties, and cognitive decline) correlates with the degree of reduced enzyme activity. Thus, patients with Gaucher disease have a severe course, whereas patients with a single mild mutation in GBA1 generally have a more benign course. Mutation carriers are also at increased risk for other PD-related disorders, including dementia with Lewy bodies, which is characterized by executive dysfunction, psychosis, and parkinsonian movement disorders, and multiple system atrophy, which has characteristic motor and cognitive impairments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further described by the accompanying drawings. These are not intended to limit the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: AAV single GEM construct of "Signal sequence" appended to "Leader+GEM F".

Figure 2: AAV four-GEM constructs with "Signal sequence" appended to "Leader+GEM F", "Leader+GEM C", "Leader+GEM D" and "Leader+GEM E".

Figure 3: Vector overview of PGRN expressing pAAV1.

Figure 4: Vector overview of PGRN expressing pAAV2.

FIG. 5 : Comparison of cell proliferation of NSC34 cells after inoculation with different combinations of AAV vectors at an MOI of 225,000.

FIG. 6 : Comparison of cell proliferation of NSC34 cells after inoculation with different combinations of AAV vectors at an MOI of 125,000.

FIG. 7 : Comparison of cell proliferation of NSC34 cells after inoculation with different combinations of AAV vectors at MOI of 225,000 and MOI of 125,000.

Figure 8: Expression of hGRN protein modules (or combinations thereof) in NSC34 cells promotes cell proliferation. Comparison of cell proliferation of NSC34 cells after viral transduction using different combinations of AAV vectors.

FIG. 9 : Expression of hGRN protein modules (or combinations thereof) in iPSC-derived motor neurons promotes cathepsin D maturation and activity.

Figure 10: Expression of hGRN protein modules (or combinations thereof) in iPSC-derived motor neurons appears to alleviate TDP-43 aggregation and accumulation.

FIG. 11 : Survival rate of NSC-34 cells after stable genomic introduction of mini-PGRN.

Figure 12: Survival of NSC-34 cells after stable genomic introduction of Grn modules (GEMs) GrnA, B, C, D, E and F or human PGRN.

FIG. 13 : Mini-PGRN CDEs and GFBs show protective activity against the toxicity of the ALS-associated molecule TDP-43 and mutant TDP-43.

Figure 14: Evaluation of the number of cells retaining a defined neuronal morphology following stress testing by serum deprivation.

Figure 15: Morphology of NSC-34 cells after stable genomic introduction of half-PGRN 14 days after serum withdrawal.

Figure 16: Morphology of NSC-34 cells after stable genomic introduction of a single Grn module (GEM) 14 days after serum withdrawal.

FIG. 17 : Length of neurite-like extensions in NSC-34 control cells, hPGRN cells, and cells stably expressing mini-PGRN GFBs and CDEs.

Detailed Description of the Drawings

Figure 1: AAV single GEM construct with "signal sequence" appended to "leader+GEM F". The signal sequence is used for native protein export of the full-length progranulin molecule, where it is naturally processed into GEM subunits at the "end" of the "leader" peptide sequence.

Figure 2: AAV four GEM constructs with "signal sequence" appended to "leader + GEM F", "leader + GEM C", "leader + GEM D" and "leader + GEM E". The signal sequence is required for native protein export of the full-length progranulin molecule, where it is naturally processed into GEM subunits at the "end" of the "leader" peptide sequence.

Figure 3: Vector overview of PGRN expressing pAAV 1. Vector overview and vector map of the example pAAV vector pAAV[Exp]-CBh>hGRN[NM_002087.4]:WPRE, a mammalian gene expression AAV vector with a CBh promoter.

Figure 4: Vector overview of PGRN expressing pAAV 2. Vector overview and vector map of the example pAAV vector pAAV[Exp]-Kan-CAG>hGRN[NM_002087.4]:WPRE3, a mammalian gene expression AAV vector with a CAG promoter.

Figure 5: Comparison of cell proliferation of NSC34 cells after inoculation with different combinations of AAV vectors. Cells were inoculated with different AAV constructs at an MOI of 225,000. Cell proliferation was analyzed by absorbance at 450 nM in a microplate reader 7, 10, and 14 days after inoculation with AAV vectors. Data points represent the mean ± SD for each condition of a single experiment performed in quadruplicate.

Figure 6: Comparison of cell proliferation of NSC34 cells after inoculation with different combinations of AAV vectors. Cells were inoculated with different AAV constructs at 125,000 MOI. Cell proliferation was analyzed by absorbance at 450 nM in a microplate reader 7, 10 and 14 days after inoculation with AAV vectors. Data points represent the mean ± SD for each condition of a single experiment performed in quadruplicate.

Figure 7: Comparison of cell proliferation of NSC34 cells after inoculation with different combinations of AAV vectors. Cells were inoculated with different AAV constructs at 125,000 MOI or 125,000 MOI. Cell proliferation was analyzed by absorbance at 450 nM in a microplate reader 7, 10 and 14 days after inoculation with AAV vectors. The data points represent the mean ± SD of the two MOI conditions of a single experiment performed in quadruplicate.

Figure 8: Expression of hGRN protein modules (or combinations thereof) in NSC34 cells promotes cell proliferation. Comparison of cell proliferation of NSC34 cells after inoculation with different combinations of AAV vectors. The data from Figures 5-7 are combined and presented relative to the level of cell proliferation after treatment with full-length PGRN.

Figure 9: Expression of hGRN protein modules (or combinations thereof) in iPSC-derived motor neurons promotes maturation and activity of cathepsin D. The absorbance of the GEM combination was normalized to full-length progranulin-infected motor neurons and plotted.

Figure 10: Expression of hGRN protein modules (or combinations thereof) in iPSC-derived motor neurons appears to mitigate TDP-43 aggregation and accumulation. The cellular concentration of TDP-43 ranged from 10,000-25,000 pg/ml (standard range: 0-75,000 pg/ml). The absorbance of the GEM combination was normalized to full-length progranulin-infected motor neurons and plotted.

Figure 11: Survival of NSC-34 cells after stable genomic introduction of a small PGRN corresponding to the amino-terminal half (GFB) and the carboxyl-terminal half (CDE) of PGRN or full-length human PGRN (hPGRN). Control cells were stably transfected with an empty vector. Cell survival was challenged by incubation in a medium containing 0% FBS. 100,000 cells were plated in each well (T0), and the number of cells was counted after 14 days. (N=3, p<0.001-***, p<0.01-**, p<0.05-*, error bars represent s.e.m.). The order of the data bars on the x-axis from left to right in the figure reflects the order of the labels presented from top to bottom in the legend.

Figure 12: Survival of NSC-34 cells after stable genomic introduction of Grn modules (GEMs) GrnA, B, C, D, E and F or human PGRN (hPGRN). Control cells were stably transfected with an empty vector. Cell survival was challenged by incubation in a medium containing 0% FBS. 100,000 cells were plated in each well (T ₀ ), and the number of cells was counted after 14 days. (N=3, p<0.001-***, p<0.01-**, p<0.05-*, error bars represent sem). The order of the data bars on the x-axis from left to right in the figure reflects the order of the labels presented from top to bottom in the legend.

Figure 13: Mini-PGRN CDE and GFB show protective activity against the toxicity of ALS-associated molecules TDP-43 and mutant TDP-43. NSC-34 cells stably expressing the full grn module or mini-PGRN were plated at 200,000 cells per well in 12-well plates containing DMEM with 10% FBS. After 24 hours, cells were transfected by liposome transfection with full-length wild-type TDP-43 or a mutant form of TDP-43 (G348C) that causes ALS, both in pCS2+, 2.5ug each. The order of the data bars on the x-axis from left to right in the figure reflects the order of the labels presented from top to bottom in the legend.

Figure 14: Number of cells that retain a clear neuronal morphology after stress testing by serum deprivation: (A) Grn modules (GEMs) A, B, C, D, E, F, and G seven days after serum withdrawal. (B) Grn modules (GEMs) A, B, C, D, E, F, and G fourteen days after serum withdrawal. (C) Small PGRNs GFB and CDE seven days after serum withdrawal (D) Small PGRNs fourteen days after serum withdrawal The order of the data bars on the x-axis from left to right in the figure reflects the order of the labels presented from top to bottom in the legend.

Figure 15: Morphology of NSC-34 cells after stable genomic introduction of half PGRN corresponding to the amino-terminal half (GFB) or carboxyl-terminal half (CDE) of PGRN 14 days after serum withdrawal. Note that in the empty vector only control, almost all cells showed evidence of apoptotic budding, detachment and rounding.

Figure 16: Morphology of NSC-34 cells following stable genomic introduction of individual Grn modules (GEMs) GrnA, GrnB, GrnC, GrnD, GrnE, GRNF and GrnG after 14 days of serum withdrawal. Note that in the empty vector only control, nearly all cells showed evidence of apoptotic budding, detachment and rounding.

Figure 17: (A) One day after serum withdrawal and (B) four days after serum withdrawal, the length of neurite-like extension in NSC-34 control cells, hPGRN cells and cells stably expressing small PGRN GFB and CDE. Note that cells stably transfected with small PGRN CDE and GFB show the same ability to promote neurite-like extension as seen in cells stably transfected with hPGRN. The order of the data bars on the x-axis from left to right in the figure reflects the order of the labels presented from top to bottom in the legend.

Example

The present invention is further described by the following examples. The examples are intended to further describe the present invention by actual examples and do not represent a limiting description of the present invention.

Progranulin is a secretory protein with important functions in a variety of physiological and pathological processes such as embryonic development, host defense, neuroprotection and wound repair. Structurally, progranulin consists of seven half-tandem repeats of the granulin/epithelialin module (GEM), several of which have been isolated as discrete 6-kDa GEM peptides, also known as granulin polypeptides. All seven human GEMs can be expressed using recombinant methods.

The present invention is based on beneficial granulins, preferably combinations of GEM/granulins, which, for example, show improved effects over full-length progranulin. Granulins and granulin combinations show beneficial properties in various in vitro models. Each of the granulins and/or granulin combinations disclosed herein will be tested in the following models. Preliminary studies have shown beneficial biological effects caused by granulins and combinations thereof, as disclosed herein.

Basic method:

The assays used to evaluate the effects of GEM and its combinations are described below:

NSC34 cell culture

Unless otherwise indicated, the NSC34 cell line was maintained in DMEM with 10% fetal bovine serum [see Cashman et al., Dev Dyn. 194: 209-21 (1992)]. For stable transfection, NSC34 cells were transfected with human granulin (pcDNA-Pgrn) or empty vector (pcDNA) using Lipofectamine (Invitrogen) and selected with G418 for one month according to the manufacturer's instructions. For example, serum deprivation assays were performed using 200,000 cells/well in 6-well plates and cultured in 4 ml RPMI (with glutamine) for 3, 6, 9, 12 and 15 days without adding or replacing fresh medium. For each time point, the average cell number was determined in 6 fields per well using an Olympus phase contrast microscope at 10X magnification.

As a further example, in the hypoxia assay, cells were plated at a density of 50,000 cells/well in 24-well plates, starved in serum-free RPMI for 24 hours, followed by addition of fresh serum-free RPMI or DMEM containing 5% serum and continued in a hypoxic chamber containing 1% O2, 5% _CO2 , balance _N2 for 72 hours. The cells were maintained in a hypoxic environment for 3 days, trypsinized and counted using a hemocytometer. For long-term culture, NSC34 cells were plated at a density of 200,000 cells/well in 6-well plates and maintained in serum-free RPMI medium. Fresh medium was provided every 10 days, and 10X magnification photos were taken at days 20 and 57 using an Olympus phase contrast microscope.

Immunofluorescence of NSC34 cells

The NSC34 cell line was cultured on glass coverslips in DMEM with 10% fetal bovine serum together with stable transfectants. The cells were fixed in 4% PFA, rinsed twice with PBST, and incubated with permeabilization buffer (PBST with 0.2% Triton X-100) for 20 minutes. After washing 3 times with PBST, the culture was post-fixed with 4% PFA for 10 minutes, followed by extensive washing. The fixed cells were incubated in PBST with 0.5% (w/v) membrane blocking reagent (GE Healthcare) for one hour, followed by the addition of sheep anti-mouse granulin (1:500 dilution, R&D Systems).

Incubation with the primary antibody was continued at 40°C overnight. The cultures were washed 3 times in PBST and then incubated with donkey anti-sheep Alexa-488 (1:200, Invitrogen) together with phalloidin-Alexa-594 conjugate (20uM) in blocking buffer for 45 minutes at room temperature. The cells were washed 3 times in PBST and then counterstained with 300 nM 4',6-diamidino-2-phenylindole (DAPI) in PBS at room temperature for 5 minutes in the dark. The cultures were washed 3 times with PBST, washed twice with _ddH2O , and then mounted on slides using Immu-mount (Thermo Fisher). Fluorescence was observed using an Axioskop 2 microscope equipped with appropriate fluorescence filters. Images were merged using Adobe Photoshop 7.0.

Apoptosis tunnel assay

NSC34 cells were plated at 200,000 cells/well on German glass photoetched coverslips (ElectronMicroscopy sciences) in 6-well plates and cultured in 4 ml RPMI (with glutamine) for six days. When fixed, the cells were washed twice in PBS and then fixed for 20 minutes using 4% PFA/PBS. After rinsing 3 times in PBST, the cells were incubated in permeabilization buffer (0.2% Triton X-100 in PBST) for 20 minutes. The cells were then post-fixed with 4% PFA/PBS for 10 minutes. After being fully washed with PBST, the cells were stored in sterile PBS at 4°C.

During treatment, cells were rinsed once with PBS and then covered with the reaction solution of fluorescein in situ death detection kit (Roche Applied Science) according to the manufacturer's instructions. Cells were incubated at 37°C for 1 hour and then rinsed twice with PBST at room temperature in the dark. After rinsing 3 times in PBST, cells were counterstained with 300nm DAPI in the dark for 5 minutes. Cells were then rinsed twice with PBST and then fixed on a slide using Immumount (ThermoFisher). Fluorescence was observed using an Axioskop2 microscope equipped with an appropriate filter, and total cells (DAPI) and apoptotic cells (FITC) were manually counted by visual inspection.

Bromodeoxyuridine (BRDU) proliferation assay

NSC34 cells were plated at 200,000/well on German glass photoetched coverslips (Electron Microscopy Sciences) in 6-well plates and cultured in 4 ml RPMI (with ammonia amide) for six days. 12 hours before fixation/treatment, BrdU labeling solution was added to each well at a concentration of 10uM (Roche Applied Sciences). During fixation, cells were washed 3 times in PBS to remove excess unintroduced BrdU and then fixed for 20 minutes using 4% PFA/PBS. After rinsing 3 times in PBST, cells were incubated in permeabilization buffer (0.2% Triton X-100 in PBST) for 20 minutes. Cells were subsequently fixed for 10 minutes with 4% PFA/PBS. After rinsing 3 times with PBST, cells were placed in 0.1M sodium borate pH 8.5 at room temperature for 2 minutes.

The cultures were incubated in PBST with 0.5% (w/v) membrane blocking reagent (GE Healthcare) for 1 hour, followed by addition of anti-BrdU Alexa-488 (1:200, Invitrogen) in blocking buffer at room temperature for 45 minutes. After rinsing 3 times in PBST, cells were counterstained with 300nm DAPI in the dark for 5 minutes. The cells were then rinsed twice with PBST, rinsed once with ddH2O, and then mounted on a slide using Immu-mount (Thermo Fisher). Fluorescence was observed using an Axioskop2 microscope equipped with appropriate filters, and total cells (DAPI) and proliferating cells (Alexa-488) were manually counted by visual inspection.

Specific examples of using the apoptosis tunnel assay or the bromodeoxyuridine (brdu) proliferation assay are not disclosed in the examples below, however, these methods represent potentially useful methods for determining the function of a GEM or GEM combination. Thus, the GEM combinations of the invention can be evaluated using these methods to display beneficial properties.

Additional methods

Cara L Ryan et al, Progranulin is expressed within motor neurons and promotes neuronal cell survival, BMC Neuroscience 2009, 10: 130 doi: 10.1186/1471-2202-10-130 describes additional in vitro methods suitable for assessing the role of progranulin.

For example, granulin provides sufficient nutrient stimulation to maintain prolonged survival of NSC-34 cells in serum-free medium. Granulin expression can prevent apoptosis of NSC-34 cells induced by serum deprivation, and exogenous granulin can increase cell survival.

Additional experimental methods for demonstrating the effects of the granulin method presented herein are disclosed in Ederle and Dormann, FEBS Letters 591 (2017) 1489-1507.

Beel et al. Molecular Neurodegeneration (2018) 13:55, https://doi.org/10.1186/s13024-018-0288-y discloses additional experimental methods for demonstrating the effects of the granulin method presented herein.

Additional methods suitable for assessing the effects of progranulin are described in Chitramuthu BP, Kay DG, Bateman A, Bennett HPJ (2017) Neurotrophic effects of progranulin in vivo inreversing motor neuron defects caused by over or under expression of TDP-43orFUS, pLoS ONE 12(3):e0174784.

For example, mutations within the GRN gene cause frontotemporal lobar degeneration (FTLD). Affected neurons display distinctive TAR DNA-binding protein 43 (TDP-43) inclusions. TDP-43 inclusions are also present in affected neurons of patients with other neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. In ALS, TDP-43 inclusions are also often immunoreactive against fusion sarcoma (FUS). Mutations within TDP-43 or FUS themselves are neuropathogenic in ALS and some cases of FTLD. Therefore, analysis of caudal primary motor neurons (MNs) grown in zebrafish embryos can be used to study the interaction of PGRN with TDP-43 and FUS in vivo. As previously reported, depletion of zebrafish PGRNA (zfPGRN-A) is associated with primary MN truncation and impaired motor function.

For example, the inventions described herein are expected to or have been demonstrated to exhibit one or more of the following effects:

Depletion of zfPGRN-A causes the growth of primary MNs to arrest at the horizontal myotome, a demarcation that divides the myotome into dorsal and ventral compartments, which is where the final destination of primary motors is allocated. Successful axon growth beyond the horizontal myotome depends in part on the formation of acetylcholine receptor clusters, and this axon growth was found to be disrupted following zfPGRN-A depletion. Granulin is considered to be a potential effective reversal of the effects of zfPGRN-A knockdown. Knockdown of both TDP-43 or FUS, as well as expression of human TDP-43 and FUS mutants, results in MN abnormalities that are expected to be reversed by co-expression of granulin. Granulin expression is expected to overcome TDP-43 and FUS neurotoxicity caused by partial loss of function or mutations in the corresponding genes, which is considered to be therapeutically relevant.

The effect of one or more granulins on the neurodegenerative phenotype of TDP-43(A315T) can be assessed. One or more granulins are expected to reduce the levels of insoluble TDP-43, and spinal cord histology shows that one or more granulins are protective against the loss of large axonal fibers in the lateral horn, which is the most severely affected fiber pool in this mouse model. Overexpression of one or more granulins is expected to significantly slow disease progression and extend median survival by approximately 130 days. We expect that one or more granulins will be effective in ameliorating mutant TDP-43-induced neurodegeneration.

The following experimental examples represent the beneficial effects of GEM treatment demonstrated by experimental methods, using the administration of GEM and GEM combinations according to the invention:

Example 1: Effect of GEM on the proliferation of NSC34 cell line:

This example aims to screen the proliferation effect of different progranulin (GRN) modules (GEM or combination thereof) in NSC34 cell line. AAV-mediated progranulin gene modules (GRN) have been tested at 225,000 and 125,000 MOI, and appropriate controls (hGRN full length and GFP) were also included.

Method summary:

In the presence of 225000MOI or 125000MOI of AAV-mediated progranulin gene modules or module combinations, NSC34 cells were seeded in 96-well plates at a density of 5.000 cells/well. Appropriate controls (hGRN, GFP and vectors) were also included. After 72h of incubation, the cell culture medium was replaced with DMEM with 1% FBS. Cell Counting Kit-8 (CCK-8) from SigmaAldrich was used to determine cell growth on days 7, 10 and 14 after infection. The assay allows for quantification of cell viability using WST-8 reagents, which are bioreduced by cell dehydrogenases to orange formazan products that are soluble in tissue culture medium. The amount of formazan produced is proportional to the number of viable cells. On the 14th day after infection, cells were stained with Hoechst. Cell nucleus images and transmission images were collected using Thermo Fisher's Cell Insight High-Content BioimagerCX7. The experiment was performed in quadruplicate.

Materials and methods:

The GEM nomenclature used in this example includes an alternative numbering scheme, as shown in the following table:

Reagents and Equipment:

-NSC34 provided by the inventor

-DMEM (Sigma-Aldrich D6429)

-FBS (Sigma-Aldrich F2442, batch BCBW6329)

- Flat bottom black 96 well plates (Becton Dickinson 353219, lot E1804340)

-Cell Insight High-Content Bioimager CX7 from Thermofisher

-Cell Counting Kit-8 (Sigma-Aldrich-96992)

Virus titer used (GC/ml):

GFP: 1.26x10 ¹²

hGRN: 1.77x10 ¹²

GEM 1: 5.65x10 ¹¹

GEM 2: 7.25x10 ¹¹

GEM 3: 1.07x10 ¹²

GEM 4: 2.26x10 ¹¹

GEM 5: 3.52x10 ¹¹

GEM 6: 6.23x10 ¹¹

GEM 7: 2.96x10 ¹¹

GEM14: 1.04x10 ¹²

GEM1374:9.35x10 ¹¹

The AAV-mediated progranulin gene module was diluted 1/10 in DMEM medium supplemented with 10% FBS to obtain the following dilution factors corresponding to 225,000 MOI and 125,000 MOI.

Actual dilution factor (uL); 1 to 10 virus stock dilution/well

The recombinant NSC34 cell line was thawed ( ^2x10 cells per T75). Cells were maintained in DMEM supplemented with 10% FBS at 37°C in a humidified 5% _CO2 atmosphere. In the presence of 225,000 MOI or 125,000 MOI of AVV-hGRN modules or combination modules, cells were plated in 96-well plates at a density of 5.000 cells per well. Cells were maintained in DMEM medium supplemented with 10% FBS at 37°C in a humidified 5% _CO2 atmosphere for 72 h. Each condition was performed in quadruplicate.

96h after inoculation, the medium was removed from the wells, and 10μl CCK-8 reagent (WST-8) + 90μl basal medium was added to each well, and the plate was incubated at 37°C. After 1 hour, the absorbance was measured at 450nm using a Synergy II microplate reader (Biotek Instruments Inc., Winooski, USA). Then, the medium containing WST-8 was removed from the wells and replaced with 200ul of the initial basal medium supplemented with 1% FBS.

10 days after inoculation, the medium was removed from the wells, and 10 μl CCK-8 reagent (WST-8) + 90 μl basal medium was added to each well, and the plate was incubated at 37°C. After 1 hour, the absorbance was measured at 450 nm using a Synergy II microplate reader (Biotek Instruments Inc., Winooski, USA). Then, the medium containing WST-8 was removed from the wells and replaced with 200 ul of the initial basal medium supplemented with 1% FBS.

14 days after inoculation, culture medium was removed from the wells, and 10 μl CCK-8 reagent (WST-8)+90 μl basal medium was added to each well, and the plate was incubated at 37 ° C. After 1 hour, absorbance was measured at 450 nm using Synergy II microplate reader (Biotek Instruments Inc., Winooski, USA). Then, Hoechst (0.5 μg/ml) was used to stain the nucleus for 30 min, and fluorescence was measured using Cell Insight High-Content Bioimager from Thermo Fisher. In order to detect Hoechst, the excitation and emission filters used were 380/10 and 460/10 nm, respectively. In addition, transmittance images were taken for each well. Image was obtained by taking 2 photos for each well using a 20x objective.

result:

Prior to adding cells, the AVV stock solution of the hGRN construct was diluted to 1/10 in DMEM with 10% serum ("stock dilution"). According to the matrix (attached method), the appropriate volume of each stock dilution was added to the appropriate wells to reach an MOI of 225,000 MOI or 125,000 MOI, respectively. The immortalized motor neuron cell line NSC-34 was harvested by trypsinization, centrifuged at 1500 rpm for 5 minutes, and then the cells were resuspended in 1 ml of complete medium (DMEM 10% FBS). To measure cell viability, 10 μl of cell solution was stained with 10 μl of trypan blue dye and counted using a hemocytometer. The cell solution was diluted to a density of 5x10 ⁴ cells/ml, and 100 μl of cell suspension containing 5000 cells was distributed in each well of a 96-well plate. After cell inoculation, the microtiter plate was incubated for 72 hours at 37°C, 5% CO ₂ , 95% air and 100% relative humidity. The medium was then removed from the wells and replaced with 200 μl of DMEM supplemented with 1% FBS. 10 days post infection, the medium was removed from the wells and replaced with 200 μl of DMEM supplemented with 1.0% FBS. The AAV-inoculated plates were monitored by microscopic observation, and cell proliferation was measured by WST8 at days 4, 10, and 14.

The inoculation efficiency of AAV vectors in this experiment was calculated by quantifying the percentage of green cells (AAV-GFP) relative to the total number of cells (total nuclei stained with Hoechst). The infection efficiency of plates A and B was 44.8% and 50.6%, respectively.

Expression of hGRN protein modules (or combinations thereof) in NSC34 cells promoted cell proliferation. NSC34 cells proliferated more efficiently in cells inoculated with 225,000 MOI of AAV containing hGRN genetic material. The growth rate relative to the negative control (GFP or uninoculated cells) became more pronounced as the number of days in culture increased, with the greatest difference occurring 14 days after inoculation.

The experimental results are shown in Figures 5 to 8 below. The results are the average of four independent replicates. For all figures: Error bars represent: +/- S.D.

Figures 5-8 show that the combination of GEM modules exhibits an enhanced effect on the enhancement of NSC34 cell proliferation. For example, in addition to GEM F+E+D+C (1+4+7+3), GEM F+E, F+B, F+C and B+C, B+E and C+E (1+4, 1+2, 1+3, 2+3, 2+4, 3+4 and 1+4+7) are all beneficial in promoting NSC34 cell proliferation. Also beneficial are the combinations 1+4+2 and 1+4+3.

As can be seen from Figure 8, which shows NSC34 proliferation relative to full-length PGRN, it is noteworthy that the GEM combination, including GEM 1 (E) with one or more of GEM2 (B), 3 (C) or 4 (E), showed higher performance than full-length PGRN compared to the other GEMs alone or in combination.

Furthermore, GEM combinations including GEM 2 (B) with one or more of GEM 3 (C) or 4 (E) showed improved performance relative to full-length PGRN and compared to other GEMs alone or in combination.

Furthermore, GEM combinations including GEM 3 (C) and GEM 4 (E) showed improved performance relative to full-length PGRN and compared to other GEMs alone or in combination.

Thus, unique combinations of GEMs, preferably those comprising double or triple GEM combinations of GEMs E, C, B and F, appear to support cell proliferation of the NSC34 motor neuron-like cell line to a greater extent than treatment with full-length PGRN or other GEMs alone or in combination.

Example 2: Effect of GEM on cathepsin D maturation in motor neuron cells with known TDP-43 mutations

This example aims to screen the cathepsin D maturation potential of different progranulin (GRN) modules (GEMs or combinations thereof) in motor neuron cell lines with known TDP-43 mutations. Cathepsins are lysosomal enzymes that are also used as sensitive markers in a variety of toxicology studies. AAV-mediated progranulin gene modules (GRNs) have been tested at 225,000 MOI, and appropriate controls (hGRN full length and GFP) were also included.

Method summary:

Human motor neurons (iPSC-derived N352S or M337V heterozygous TDP-43 mutations) are derived from genetically modified normal iPSC lines carrying heterozygous N352S or M337V mutations in the TDP-43 gene. iXCells ^™ hiPSC-derived motor neurons express typical markers of motor neurons, such as HB9 (MNX1), ISL1, CHAT, with a purity greater than 85%. Most cells express high levels of HB9 and ISL-1 after thawing, and high levels of CHAT and MAP2 after 5-7 days. In the presence of an AAV-mediated progranulin gene module or module combination at 250,000 MOI, induced pluripotent stem cells that are ultimately differentiated into motor neurons are seeded in 96-well plates at a density of 10,000 cells/well. Appropriate controls (hGRN, GFP, and carriers) are also included. Seven days after infection, a cathepsin-D activity assay from RayBiotech (Cat#: 68AT-CathD-S100) was used to determine whether individual GEMs or GEM combinations enhanced the efficiency of TDP-43 mutant motor neurons to cleave the preferred cathepsin-D substrate sequence GKPILFFRLK(Dnp)-DR-NH2 labeled with MCA. Quantification was performed using a fluorometer or fluorescence plate reader at Ex/Em=328/460nm.

Materials and methods:

The GEM nomenclature used in this example includes an alternative numbering scheme, as shown in the following table:

Reagents and Equipment:

-iXCell human motor neurons (iPSC-derived TDP-43 mutants N352S, HET): 40HU-102-2M

-iXCell Human Motor Neurons (iPSC-derived TDP-43 mutants Q331K, HET): 40HU-103-2M

-iXCell Motor Neuron Maintenance Medium (Cat# MD-0022)

-RayBiotech Cathepsin D Activity Assay Kit: 68AT-CathD-S100

- Flat bottom black 96 well plates (Becton Dickinson 353219, lot E1804340)

Virus titer used (GC/ml):

GFP: 1.26x10 ¹²

hGRN: 4.37x10 ¹³

GEM 1: 5.65x10 ¹¹

GEM 2: 7.25x10 ¹¹

GEM 3: 1.07x10 ¹²

GEM 4: 2.26x10 ¹¹

GEM 5: 3.52x10 ¹¹

GEM 6: 6.23x10 ¹¹

GEM 7: 2.96x10 ¹¹

GEM14: 1.04x10 ¹²

GEM1374:9.35x10 ¹¹

The AAV-mediated progranulin gene module was diluted 1/10 in motor neuron maintenance medium to obtain the following dilution factors corresponding to an MOI of 250,000.

Actual dilution factor (uL): 1 to 10 virus stock dilution/well

MOI 250000 GFP 20 HkDJ 1 GEM 1 45 GEM 2 35 GEM 3 twenty four GEM 4 111 GEM 5 72 GEM 6 41 GEM 7 85 GEM14 25 GEM1374 27

The iPSC-derived motor neuron cells were thawed (1x106 cells per 96-well plate). The cells were maintained in motor neuron maintenance medium at 37°C in a humidified 5% CO2 atmosphere. The cells were plated in 96-well plates at a density of 10,000 cells per well. After 24 hours, a 1 to 10 dilution of the virus was added to the final 250,000 MOI AVV-hGRN module or combination module. At 37°C in a humidified 5% CO2 atmosphere, the cells were maintained in motor neuron maintenance medium for 72h.

Five days after inoculation, the culture medium was removed from the wells, and 10 μl of CCK-8 reagent (WST-8) + 90 μl of basal medium was added to each well, and the plate was incubated at 37° C. After 1 hour, the absorbance was measured at 450 nm using a Synergy II microplate reader (Biotek Instruments Inc., Winooski, USA). Then, the culture medium containing WST-8 was removed from the wells and the cathepsin D assay was performed.

Determination procedure:

1. Collect cells (1-2 x 10 ⁶ ) by centrifugation.

2. Lyse cells in 200 μl of chilled CD40 cell lysis buffer. Incubate cells on ice for 10 min.

3. Centrifuge at high speed for 5 min in a microcentrifuge and transfer the supernatant to a new tube. For each measurement, add 5-50 μl of cell lysate to a 96-well plate.

4. Adjust the volume of CD reaction buffer to 50 μl for each sample.

5. Prepare the master assay mix for each assay. Each assay requires: 50 μl reaction buffer + 2 μl CD substrate. Mix well.

6. Add 52 μl of the stock solution to each well. Stir well.

7. Incubate at 37°C for 1-2 hours.

8. Read the samples in a fluorimeter equipped with a 328 nm excitation filter and a 460 nm emission filter.

Fold increase in cathepsin D activity can be determined by comparing relative fluorescence units (RFU) per million cells, or RFU per microgram of protein in a cell lysate sample, or the fold increase in RFU of treated versus untreated control or negative control samples.

result:

The absorbance of the GEM combination was normalized to that of full-length progranulin infected motor neurons and plotted. Calculated values above 1.00 were interpreted as the ability of the GEM combination to increase the maturation of cathepsin D, thereby releasing more fluorescent substrate, than in cells expressing full-length progranulin. Calculated values below 1.00 were interpreted as having no effect or a negative effect on the ability of motor neurons to promote maturation of pro-cathepsin D into fully mature and functional cathepsin D protein.

Expression of hGRN protein modules (or combinations thereof) in iPSC-derived motor neurons promotes maturation and activity of cathepsin D. Relative to full-length progranulin AAV-infected cells, cathepsin D substrate cleavage rates show variability due to GEM combinations and TDP-43 mutations.

The data is shown in Figure 9.

As can be seen from Figure 9, which shows the results of the cathepsin D maturation assay relative to full-length PGRN, it is noteworthy that the GEM combinations including 1+4, 1+5, 1+7 (F+E, D or G) showed improved performance relative to full-length PGRN and compared to other GEMs alone or in combination.

Furthermore, GEM combinations including GEM 2 with one or more of 3, 4, 5, 6 or 7 (GEM B with C, E, G, A or D) showed improved performance relative to full-length PGRN and compared to other GEMs alone or in combination.

Furthermore, GEM combinations including GEM 3 with one or more of 3, 4, 5, 6 or 7 (GEM C with E, G, A or D) showed improved performance relative to full-length PGRN and compared to other GEMs alone or in combination.

Furthermore, GEM combinations including GEM 4 with one or more of 5, 6 or 7 (GEM E with G, A or D) showed improved performance relative to full-length PGRN and compared to other GEMs alone or in combination.

Furthermore, GEM combinations including GEM 5 with one or more of 6 or 7 (GEM G with A or D) showed improved performance relative to full-length PGRN and compared to other GEMs alone or in combination. GEM 6+7 and 7 alone also showed improvements.

Under these experimental conditions, the peptide module combinations showing the greatest improvement appeared to be 2+7, 3+7, 4+7, 5+7, 6+7 and GEM 7 alone (B+D, C+D, E+D, G+D, A+D and GEM D), which all showed enhanced cathepsin D action in motor neuron cells. Combinations with GEM 4, 1+4 and 1+4+7 (GEM E combination and F+E+D) also showed an enhancing effect on cathepsin D maturation levels.

Example 3: Effect of GEM on TDP43 levels in motor neuron cells with known TDP43 mutations

This embodiment aims to screen different progranulin (GRN) modules (GEM or its combination) to change/reduce the potential of TDP43 protein accumulation in motor neuron cell lines with known TDP43 mutations. Human TDP43 is an RNA binding protein involved in each step of RNA biosynthesis and processing. Abnormal RNA processing, cell compartmentalization and protein degradation are related to mutations of TDP43 protein and are associated with various nervous system diseases. AAV-mediated progranulin gene module (GRN) has been tested at 225000 MOI, and also includes appropriate controls (hGRN full length and GFP).

Method summary:

Human motor neurons (iPSC-derived N352S or M337V heterozygous TDP-43 mutations) are derived from genetically modified normal iPSC lines carrying heterozygous N352S or M337V mutations in the TDP-43 gene. iXCells ^™ hiPSC-derived motor neurons express typical markers of motor neurons, such as HB9 (MNX1), ISL1, CHAT, with a purity greater than 85%. Most cells express high levels of HB9 and ISL-1 after thawing, and high levels of CHAT and MAP2 after 5-7 days. In the presence of an AAV-mediated progranulin gene module or module combination at 250,000 MOI, induced pluripotent stem cells that are ultimately differentiated into motor neurons are seeded in 96-well plates at a density of 10,000 cells/well. Appropriate controls (hGRN, GFP, and carriers) are also included. Seven days after infection, a cathepsin-D activity assay from RayBiotech (Cat#: 68AT-CathD-S100) was used to determine whether individual GEMs or GEM combinations enhanced the efficiency of TDP-43 mutant motor neurons to cleave the preferred cathepsin-D substrate sequence GKPILFFRLK(Dnp)-DR-NH2 labeled with MCA. Quantification was performed using a fluorometer or fluorescence plate reader at Ex/Em=328/460nm.

Materials and methods:

Reagents and Equipment:

-iXCell human motor neurons (iPSC-derived TDP-43 mutants N352S, HET): 40HU-102-2M

-iXCell Human Motor Neurons (iPSC-derived TDP-43 mutants Q331K, HET): 40HU-103-2M

-iXCell Motor Neuron Maintenance Medium (Cat# MD-0022)

-Abcam Human TDP43 SimpleStep ELISA Kit (TARDBP): ab282880

- Flat bottom black 96 well plates (Becton Dickinson 353219, lot E1804340)

Virus titer used (GC/ml):

GFP: 1.26x10 ¹²

hGRN: 4.37x10 ¹³

GEM 1: 5.65x10 ¹¹

GEM 2: 7.25x10 ¹¹

GEM 3: 1.07x10 ¹²

GEM 4: 2.26x10 ¹¹

GEM 5: 3.52x10 ¹¹

GEM 6: 6.23x10 ¹¹

GEM 7: 2.96x10 ¹¹

GEM14: 1.04x10 ¹²

GEM1374:9.35x10 ¹¹

The AAV-mediated progranulin gene module was diluted 1/10 in motor neuron maintenance medium to obtain the following dilution factors corresponding to an MOI of 250,000.

Actual dilution factor (uL): 1 to 10 virus stock dilution/well

The iPSC-derived motor neuron cells were thawed (1x106 cells per 96-well plate). At 37°C in a humidified 5% CO2 atmosphere, the cells were maintained in motor neuron maintenance medium. The cells were plated in 96-well plates at a density of 10.000 cells per well. After 24 hours, a 1 to 10 dilution of the virus was added to the final 250000 MOI AVV-hGRN module or combination module. At 37°C in a humidified 5% CO2 atmosphere, the cells were maintained in motor neuron maintenance medium for 72h.

Five days after inoculation, the culture medium was removed from the wells, and 10 μl of CCK-8 reagent (WST-8) + 90 μl of basal medium was added to each well, and the plate was incubated at 37° C. After 1 hour, the absorbance was measured at 450 nm using a Synergy II microplate reader (Biotek Instruments Inc., Winooski, USA). Then, the culture medium containing WST-8 was removed from the wells and the cathepsin D assay was performed.

Determination procedure:

1. Prepare all reagents, cell samples and standards according to the instructions

2. Add 50 μL of standard or sample to the corresponding wells

3. Add 50 μL of antibody mixture to all wells

4. Incubate at room temperature for 1 hour

5. Aspirate dry and wash each well 3 times with 350 μL 1X Wash Buffer PT

6. Add 100 μL of TMB developing solution to each well and incubate for 10 minutes.

7. Add 100 μL of stop solution and read OD at 450 nm

result:

The cell concentration range of TDP-43 is 10,000-25,000pg/ml (standard range: 0-75,000pg/ml). The absorbance of the GEM combination is normalized and plotted for motor neurons infected with full-length progranulin. Calculated values below 1.00 are interpreted as GEM combinations being able to reduce TDP-43 concentrations more than cells expressing full-length progranulin. Calculated values above 1.00 are interpreted as having no effect or a negative impact on the ability of motor neurons to process and resolve TDP-43 protein diseases.

Expression of hGRN protein modules (or combinations thereof) in iPSC-derived motor neurons appears to alleviate TDP-43 aggregation and accumulation. TDP-43 accumulation rates relative to full-length progranulin show some variability in GEM combinations and TDP-43 mutations.

The data is shown in Figure 10.

As can be seen from Figure 10, which shows the TDP-43 accumulation results relative to full-length PGRN, it is noteworthy that the GEM combinations including 1+4, 1+5, 1+6, 1+7 (F+E, D, A or G) showed improved performance relative to full-length PGRN and compared to other GEMs alone or in combination.

Furthermore, the GEM combination including GEM2 and 6 (GEM B and A) showed improved performance relative to full-length PGRN and compared to other GEMs alone or in combination.

Under these experimental conditions, the combinations of peptide modules 1+4, 1+5, 1+6 and 1+7 (F+E, F+A and F+D) appear to show the greatest enhancement of the ability of motor neuron cells to properly clear TDP-43, even in the presence of known TDP-43 mutations. 1+4+6 and 1+4+7 (F+E+A and F+E+D) also showed an enhancement effect on reducing TDP-43 protein levels.

Example 4: Progranulin after GEM treatment in motor neuron cells with known TDP-43 mutations Expression level

This example aims to screen different progranulin (GRN) modules (or combinations thereof) for any potential effect on the total expression of full-length progranulin in a motor neuron cell line with known TDP-43 mutations. AAV-mediated progranulin gene modules (GRN) were tested at an MOI of 250,000 and appropriate controls (hGRN full length and GFP) were included.

Method summary:

Human motor neurons (iPSC-derived N352S or M337V heterozygous TDP-43 mutations) are derived from genetically modified normal iPSC lines carrying heterozygous N352S or M337V mutations in the TDP-43 gene. iXCellsTMhiPSC-derived motor neurons express typical markers of motor neurons, such as HB9 (MNX1), ISL1, CHAT, with a purity greater than 85%. Most cells express high levels of HB9 and ISL-1 after thawing, and high levels of CHAT and MAP2 after 5-7 days. In the presence of an AAV-mediated progranulin gene module or module combination of 250,000 MOI, induced pluripotent stem cells that are ultimately differentiated into motor neurons are seeded in 96-well plates at a density of 10,000 cells/well. Appropriate controls (hGRN, GFP, and carriers) are also included.

Determination procedure:

The assay is a sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of human progranulin in biological fluids. A polyclonal antibody specific for progranulin is pre-coated on a 96-well microtiter plate. Standards and samples are pipetted into the wells to bind to the coated antibody. After extensive washing to remove unbound compounds, progranulin is recognized by the addition of a biotinylated polyclonal antibody specific for progranulin (detection antibody). After removal of excess biotinylated antibody, HRP-labeled streptavidin (STREP-HRP) is added. After a final wash, peroxidase activity is quantified using the substrate 3,3',5,5'-tetramethylbenzidine (TMB). The intensity of the color reaction is measured at 450 nm after acidification and is proportional to the concentration of progranulin in the sample.

It is expected that a strong signal will be generated in the full-length progranulin AAV wells. The inventors speculate that the expression of the hGRN protein module (GEM or combination thereof) may lead to increased PGRN levels in the treated wells. The GEM (or combination thereof) may "release" the full-length progranulin, thereby generating a stronger signal because the GEM is performing the functions originally required to process the endogenous full-length progranulin.

Example 5: Effects of GEM on Neuroinflammation

Elevated neuroinflammation is a pathological hallmark of neurodegenerative diseases. It is known that full-length PGRN exhibits anti-neuroinflammatory properties. In contrast, excessive neuroinflammation was observed in animals with PGRN deficiency, where increased levels of activated astrocytes and microglia were observed with age. This example aims to screen the potential of different progranulin (GRN) modules (GEMs or combinations thereof) for anti-neuroinflammatory effects, as observed for full-length progranulin.

The lead candidate individual GEMs or GEM combinations are evaluated for their ability to affect pro-inflammatory and anti-inflammatory cytokine production in a human microglial cell line, for example, in an assay selected from (i) a cell survival assay, (ii) an assay to promote a mature neuronal phenotype, and/or (iii) an assay to measure reduction in toxic TDP-43. The effects of the candidate GEMs/GEM combinations on cytokine production are evaluated in two culture conditions, namely in the absence of lipopolysaccharide (LPS) activation and in the presence of lipopolysaccharide (LPS) activation.

Method summary:

The human microglial cell line HMC3 (ATCC, CRL-3304) was infected with an AAV-9 viral vector encoding a candidate individual GEM or a combination of GEMs. Three days after infection, the cells were subcultured and exposed to LPS stimulation or not (0.5 mg/ml, 24 hours) to activate microglia. Twenty-four hours later, the V-PLEX pro-inflammatory group 1 human kit (Mesoscale K15049D; alternative kits can be used) was used to analyze the content of a group of pro-inflammatory cytokines and anti-inflammatory cytokines in the cell culture supernatant. The kit can quantify both a series of pro-inflammatory cytokines and anti-inflammatory cytokines. Therefore, IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNF-α can be quantified.

The effects of candidate GEM/GEM combinations on cytokine production were compared to that observed in cell culture supernatants from HMC3 cells expressing full-length PGRN (AAV-9PGRN infected) as a positive control or GFP expressing and uninfected HMC3 cells as a negative control.

The inventors hypothesize that expression of hGRN protein modules (GEMs or combinations thereof) exhibits beneficial anti-inflammatory effects at levels similar to or improved upon treatment with full-length PGRN.

Example 6: NSC-34 cells after stable genomic introduction of GEM, mini-PGRN and full-length human PGRN (hPGRN) Cell survival and maintenance of neuronal morphology under serum deprivation stress

The present example aims to screen the survival enhancing effect of stable genomic integration of different progranulin (GRN) modules (GEMs or combinations thereof) or mini-PGRNs (corresponding to the amino-terminal half (GFB) or the carboxyl-terminal half (CDE) of the full-length human PGRN (hPGRN)) in the NSC34 cell line.

Evaluation of NSC-34 cells stably expressing human progranulin (hPGRN), mini-PGRN containing the N-terminal progranulin module GFB or the C-terminal progranulin module CDE, or individual GRN modules A, B, C, D, E, F, and G

(A): Survival rate under serum deprivation stress,

(B): Survival rate after expression of ALS-related molecule TDP-43 wild type and mutant TDP-43 (G348C),

(D): Maintenance of neuronal morphology during serum deprivation stress, and

(C): The rate of neurite extension during serum deprivation stress.

method:

Carrier:

A single GEM and a small PGRN CDE and GFB were inserted (hPGRN-SP-pcDNA3.1/V5-His TOPO) into a modified pcDNA3.1/V5-His TOPO (A. Bateman and B Chitramuthu) designed to include a human PGRN secretion signal sequence. The plasmid was selected, purified, and sequenced to ensure the fidelity of the sequence to confirm that the insert was in frame and that the hPGRN secretion signal peptide was inserted into the amino terminus of the final protein product in the correct orientation. The secretion signal peptide enables the protein to be secreted and transported to the lysosome.

Cell culture, transfection and validation:

NSC-34 cells were used as described above. NSC-34 cells were maintained in DMEM with 10% fetal bovine serum (FBS). Stable transfectants expressing GEM grnA, grnB, grnC, grnD, grnE, grnF, grnG, mini-pgrn CDE and mini-pgrn GFB were generated by transfecting empty vector control transfectants with GEM (in sp-pcDNA3.1 V5-Topo-GEM) or sp-pcDNA3.1 V5-Topo. Cells were transfected with Lipofectamine (Invitrogen) and selected for 4 to 6 weeks with G418 (400 μg/ml) according to the manufacturer's instructions. In order to prevent phenotypic drift, the stock solution of the original transfectant was frozen in liquid nitrogen and revived regularly. Expression was confirmed by RT-PCR using total RNA isolated using Trizol reagent (Invitrogen). cDNA synthesis was performed using Revert-aid reverse transcriptase (Thermo Scientific). GEM-specific primer sets were designed to amplify specific products.

Cell survival bioassay:

NSC 34 cells expressing the full grn module or small PGRN were plated in 6-well plates containing DMEM with 10% FBS at 100,000 cells per well. After 24 hours, the culture medium was replaced with DMEM containing 0% FBS. The cells were trypsinized and counted using the trypan blue dye exclusion assay on the 14th day to distinguish live cells from dead cells. GraphPad software (GraphPad Prism Software Inc., San Diego, CA) was used to determine the statistical significance between the experimental groups by one-way analysis of variance followed by Tukey's multiple comparison test (p<0.001-***, p<0.01-**, p<0.05-*). Error bars represent s.e.m.

Cell Survival Bioassay - TDP-43 Challenge:

NSC-34 cells stably expressing the full grn module or mini-PGRN were plated at 200,000 cells per well in 12-well plates containing DMEM with 10% FBS. After 24 hours, cells were transfected by lipofection with full-length wild-type TDP-43 or a mutant form of TDP-43 that causes ALS (G348C), both in pCS2+, 2.5ug each. Control cells were mock-transfected with a reagent lacking DNA. Cells were propagated in 10% serum for four days, trypsinized, and viable cell counts were assessed by trypan blue exclusion test.

morphology:

NSC-34 cells expressing the full grn module or small PGRN were plated at 100,000 cells per well in 12-well plates containing DMEM with 10% FBS. After 24 hours, the culture medium was replaced with DMEM containing 0% FBS. In order to evaluate the maintenance of cell neuronal morphology under stress conditions, the cultures were monitored on the 7th and 14th days in serum-free medium, and photos were taken using an inverted phase contrast microscope. Neuronal morphology was assessed as scattered cell bodies (not round cells) with clearly visible neurites extending at least twice the length of the cell body along its longest axis.

To evaluate the rate of neurite extension, we tested the two best performing constructs described above and assessed their average neurite extension length after 1 and 4 days in serum-free medium compared to CTL cells stably transfected with empty vector and hPGRN-expressing cells. Cells were photographed on an inverted microscope and extension length was assessed using the ImageJ program from the FIJI open source image processing package.

result:

A. Serum deprivation stress challenge:

The survival of NSC-34 cells was evaluated after stable genomic introduction of a mini-PGRN corresponding to the amino-terminal half (GFB) and carboxyl-terminal half (CDE) of PGRN or full-length human PGRN (hPGRN). Control cells were stably transfected with an empty vector. Cell survival was challenged by incubation in a medium containing 0% FBS. 100,000 cells were plated in each well (T0) and the number of cells was counted after 14 days.

As can be seen in Figure 11, the protection provided by mini-PGRN GFB and CDE against serum deprivation stress is close to that provided by full-length hPGRN. Since both the N-terminal mini-PGRN GFB and the C-terminal mini-PGRN CDE provided protection, it is clear that the activity is not limited to a single locus on the full-length PGRN, but is distributed between the N-terminal and C-terminal portions of PGRN.

As can be seen in Figure 12, among the GEM modules (GEMs), E and F provided the strongest protection, indicating that the protection activity detected in CDE may be concentrated in module E, and that in GFB may be concentrated in module F. Individual modules provided less protection than the GEM combination, such as the mini-PGRN used in this experiment (see Figure 11 above), suggesting that the activity of E and F is enhanced by the presence of other (possibly less active) modules in the mini-PGRN (G, F in GFB and D, E in CDE).

B. Survival: TDP-43 cytotoxicity challenge:

NSC-34 cells stably expressing the full grn module or mini-PGRN were plated at 200,000 cells per well in 12-well plates containing DMEM with 10% FBS. 24 hours later, cells were transfected by lipofection with full-length wild-type TDP-43 or a mutant form of TDP-43 that causes ALS (G348C), both in pCS2+, 2.5ug each.

As can be seen in Figure 13, CDE and GFB small PGRNs both showed protection against ALS-related TDP-43 toxicity for WT (i.e., sporadic ALS) and G348C (mutant ALS), close to the protection provided by full-length hPGRN. Small PGRNs CDE and GFB showed protective activity against the toxicity of ALS-related molecules TDP-43 and mutant TDP-43.

C. Maintenance of neuronal morphology after serum deprivation stress:

After the stress test by serum deprivation, the cell number of retaining clear neuronal morphology was evaluated. As can be seen from Figure 14, single Grn modules (GEM) G, F, B and E show the trend of improving morphological maintenance (inset B). Both small PGRN GFB and CDE show that the maintenance of neuronal morphology is improved after the serum deprivation stress of 14 days, which is no different from hPGRN.

Additional data are shown in Figures 15 and 16 , demonstrating the morphology of NSC-34 cells after stable genomic introduction of half PGRN 14 days after serum withdrawal, and the morphology of NSC-34 cells after stable genomic introduction of individual Grn modules (GEMs) GrnA, GrnB, GrnC, GrnD, GrnE, GrnF, and GrnG 14 days after serum withdrawal.

D. Neurite extension rate:

The best performing constructs A to C were tested for their ability to promote neurite extension compared to empty vector NSC-43 control cells and NSC-34 hPGRN expressing cells in serum-depleted medium. Assays were performed on days 1 and 4, both days before CTL cells initiate extensive apoptosis.

As can be seen in FIG. 17 , cells stably transfected with mini-PGRN CDE and GFB showed equivalent ability to promote neurite-like extension as that seen in cells stably transfected with hPGRN.

Additional GEMs and GEM combinations of the present invention were tested in the experiments of Examples 1 to 6. The inventors hypothesized that the effects observed in the examples disclosed herein could be reproduced for the same and additional GEM combinations of the present invention.

Sequence Listing

<110> Alpha Cognition Inc.

<120> Granulin/epithelialin modules and combinations thereof for treating neurodegenerative diseases

<130> PPI23172654DE

<150> US63/191255

<151> 2021-05-20

<150> US63/309118

<151> 2022-02-11

<160> 39

<170> PatentIn version 3.5

<210> 1

<211> 593

<212> PRT

<213> Homo sapiens

<400> 1

Met Trp Thr Leu Val Ser Trp Val Ala Leu Thr Ala Gly Leu Val Ala

1 5 10 15

Gly Thr Arg Cys Pro Asp Gly Gln Phe Cys Pro Val Ala Cys Cys Leu

20 25 30

Asp Pro Gly Gly Ala Ser Tyr Ser Cys Cys Arg Pro Leu Leu Asp Lys

35 40 45

Trp Pro Thr Thr Leu Ser Arg His Leu Gly Gly Pro Cys Gln Val Asp

50 55 60

Ala His Cys Ser Ala Gly His Ser Cys Ile Phe Thr Val Ser Gly Thr

65 70 75 80

Ser Ser Cys Cys Pro Phe Pro Glu Ala Val Ala Cys Gly Asp Gly His

85 90 95

His Cys Cys Pro Arg Gly Phe His Cys Ser Ala Asp Gly Arg Ser Cys

100 105 110

Phe Gln Arg Ser Gly Asn Asn Ser Val Gly Ala Ile Gln Cys Pro Asp

115 120 125

Ser Gln Phe Glu Cys Pro Asp Phe Ser Thr Cys Cys Val Met Val Asp

130 135 140

Gly Ser Trp Gly Cys Cys Pro Met Pro Gln Ala Ser Cys Cys Glu Asp

145 150 155 160

Arg Val His Cys Cys Pro His Gly Ala Phe Cys Asp Leu Val His Thr

165 170 175

Arg Cys Ile Thr Pro Thr Gly Thr His Pro Leu Ala Lys Lys Leu Pro

180 185 190

Ala Gln Arg Thr Asn Arg Ala Val Ala Leu Ser Ser Ser Val Met Cys

195 200 205

Pro Asp Ala Arg Ser Arg Cys Pro Asp Gly Ser Thr Cys Cys Glu Leu

210 215 220

Pro Ser Gly Lys Tyr Gly Cys Cys Pro Met Pro Asn Ala Thr Cys Cys

225 230 235 240

Ser Asp His Leu His Cys Cys Pro Gln Asp Thr Val Cys Asp Leu Ile

245 250 255

Gln Ser Lys Cys Leu Ser Lys Glu Asn Ala Thr Thr Asp Leu Leu Thr

260 265 270

Lys Leu Pro Ala His Thr Val Gly Asp Val Lys Cys Asp Met Glu Val

275 280 285

Ser Cys Pro Asp Gly Tyr Thr Cys Cys Arg Leu Gln Ser Gly Ala Trp

290 295 300

Gly Cys Cys Pro Phe Thr Gln Ala Val Cys Cys Glu Asp His Ile His

305 310 315 320

Cys Cys Pro Ala Gly Phe Thr Cys Asp Thr Gln Lys Gly Thr Cys Glu

325 330 335

Gln Gly Pro His Gln Val Pro Trp Met Glu Lys Ala Pro Ala His Leu

340 345 350

Ser Leu Pro Asp Pro Gln Ala Leu Lys Arg Asp Val Pro Cys Asp Asn

355 360 365

Val Ser Ser Cys Pro Ser Ser Asp Thr Cys Cys Gln Leu Thr Ser Gly

370 375 380

Glu Trp Gly Cys Cys Pro Ile Pro Glu Ala Val Cys Cys Ser Asp His

385 390 395 400

Gln His Cys Cys Pro Gln Gly Tyr Thr Cys Val Ala Glu Gly Gln Cys

405 410 415

Gln Arg Gly Ser Glu Ile Val Ala Gly Leu Glu Lys Met Pro Ala Arg

420 425 430

Arg Ala Ser Leu Ser His Pro Arg Asp Ile Gly Cys Asp Gln His Thr

435 440 445

Ser Cys Pro Val Gly Gln Thr Cys Cys Pro Ser Leu Gly Gly Ser Trp

450 455 460

Ala Cys Cys Gln Leu Pro His Ala Val Cys Cys Glu Asp Arg Gln His

465 470 475 480

Cys Cys Pro Ala Gly Tyr Thr Cys Asn Val Lys Ala Arg Ser Cys Glu

485 490 495

Lys Glu Val Val Ser Ala Gln Pro Ala Thr Phe Leu Ala Arg Ser Pro

500 505 510

His Val Gly Val Lys Asp Val Glu Cys Gly Glu Gly His Phe Cys His

515 520 525

Asp Asn Gln Thr Cys Cys Arg Asp Asn Arg Gln Gly Trp Ala Cys Cys

530 535 540

Pro Tyr Arg Gln Gly Val Cys Cys Ala Asp Arg Arg His Cys Cys Pro

545 550 555 560

Ala Gly Phe Arg Cys Ala Ala Arg Gly Thr Lys Cys Leu Arg Arg Glu

565 570 575

Ala Pro Arg Trp Asp Ala Pro Leu Arg Asp Pro Ala Leu Arg Gln Leu

580 585 590

Leu

<210> 2

<211> 57

<212> PRT

<213> Homo sapiens

<400> 2

Ala Ile Gln Cys Pro Asp Ser Gln Phe Glu Cys Pro Asp Phe Ser Thr

1 5 10 15

Cys Cys Val Met Val Asp Gly Ser Trp Gly Cys Cys Pro Met Pro Gln

20 25 30

Ala Ser Cys Cys Glu Asp Arg Val His Cys Cys Pro His Gly Ala Phe

35 40 45

Cys Asp Leu Val His Thr Arg Cys Ile

50 55

<210> 3

<211> 56

<212> PRT

<213> Homo sapiens

<400> 3

Val Met Cys Pro Asp Ala Arg Ser Arg Cys Pro Asp Gly Ser Thr Cys

1 5 10 15

Cys Glu Leu Pro Ser Gly Lys Tyr Gly Cys Cys Pro Met Pro Asn Ala

20 25 30

Thr Cys Cys Ser Asp His Leu His Cys Cys Pro Gln Asp Thr Val Cys

35 40 45

Asp Leu Ile Gln Ser Lys Cys Leu

50 55

<210> 4

<211> 54

<212> PRT

<213> Homo sapiens

<400> 4

Val Pro Cys Asp Asn Val Ser Ser Cys Pro Ser Ser Asp Thr Cys Cys

1 5 10 15

Gln Leu Thr Ser Gly Glu Trp Gly Cys Cys Pro Ile Pro Glu Ala Val

20 25 30

Cys Cys Ser Asp His Gln His Cys Cys Pro Gln Gly Tyr Thr Cys Val

35 40 45

Ala Glu Gly Gln Cys Gln

50

<210> 5

<211> 56

<212> PRT

<213> Homo sapiens

<400> 5

Asp Val Glu Cys Gly Glu Gly His Phe Cys His Asp Asn Gln Thr Cys

1 5 10 15

Cys Arg Asp Asn Arg Gln Gly Trp Ala Cys Cys Pro Tyr Arg Gln Gly

20 25 30

Val Cys Cys Ala Asp Arg Arg His Cys Cys Pro Ala Gly Phe Arg Cys

35 40 45

Ala Ala Arg Gly Thr Lys Cys Leu

50 55

<210> 6

<211> 56

<212> PRT

<213> Homo sapiens

<400> 6

Gly Gly Pro Cys Gln Val Asp Ala His Cys Ser Ala Gly His Ser Cys

1 5 10 15

Ile Phe Thr Val Ser Gly Thr Ser Ser Ser Cys Cys Pro Phe Pro Glu Ala

20 25 30

Val Ala Cys Gly Asp Gly His His Cys Cys Pro Arg Gly Phe His Cys

35 40 45

Ser Ala Asp Gly Arg Ser Cys Phe

50 55

<210> 7

<211> 57

<212> PRT

<213> Homo sapiens

<400> 7

Asp Val Lys Cys Asp Met Glu Val Ser Cys Pro Asp Gly Tyr Thr Cys

1 5 10 15

Cys Arg Leu Gln Ser Gly Ala Trp Gly Cys Cys Pro Phe Thr Gln Ala

20 25 30

Val Cys Cys Glu Asp His Ile His Cys Cys Pro Ala Gly Phe Thr Cys

35 40 45

Asp Thr Gln Lys Gly Thr Cys Glu Gln

50 55

<210> 8

<211> 56

<212> PRT

<213> Homo sapiens

<400> 8

Asp Ile Gly Cys Asp Gln His Thr Ser Cys Pro Val Gly Gln Thr Cys

1 5 10 15

Cys Pro Ser Leu Gly Gly Ser Trp Ala Cys Cys Gln Leu Pro His Ala

20 25 30

Val Cys Cys Glu Asp Arg Gln His Cys Cys Pro Ala Gly Tyr Thr Cys

35 40 45

Asn Val Lys Ala Arg Ser Cys Glu

50 55

<210> 9

<211> 2130

<212> DNA

<213> Homo sapiens

<400> 9

gctgctgccc aaggaccgcg gagtcggacg caggcagacc atgtggaccc tggtgagctg 60

ggtggcctta acagcagggc tggtggctgg aacgcggtgc ccagatggtc agttctgccc 120

tgtggcctgc tgcctggacc ccggaggagc cagctacagc tgctgccgtc cccttctgga 180

caaatggccc acaacactga gcaggcatct gggtggcccc tgccaggttg atgcccactg 240

ctctgccggc cactcctgca tctttaccgt ctcagggact tccagttgct gccccttccc 300

agaggccgtg gcatgcgggg atggccatca ctgctgccca cggggcttcc actgcagtgc 360

agacgggcga tcctgcttcc aaagatcagg taacaactcc gtgggtgcca tccagtgccc 420

tgatagtcag ttcgaatgcc cggacttctc cacgtgctgt gttatggtcg atggctcctg 480

ggggtgctgc cccatgcccc aggcttcctg ctgtgaagac agggtgcact gctgtccgca 540

cggtgccttc tgcgacctgg ttcacacccg ctgcatcaca cccacgggca cccaccccct 600

ggcaaagaag ctccctgccc agaggactaa cagggcagtg gccttgtcca gctcggtcat 660

gtgtccggac gcacggtccc ggtgccctga tggttctacc tgctgtgagc tgcccagtgg 720

gaagtatggc tgctgcccaa tgcccaacgc cacctgctgc tccgatcacc tgcactgctg 780

cccccaagac actgtgtgtg acctgatcca gagtaagtgc ctctccaagg agaacgctac 840

cacggacctc ctcactaagc tgcctgcgca cacagtgggg gatgtgaaat gtgacatgga 900

ggtgagctgc ccagatggct atacctgctg ccgtctacag tcgggggcct ggggctgctg 960

cccttttacc caggctgtgt gctgtgagga ccacatacac tgctgtcccg cggggtttac 1020

gtgtgacacg cagaagggta cctgtgaaca ggggccccac caggtgccct ggatggagaa 1080

ggccccagct cacctcagcc tgccagaccc acaagccttg aagagagatg tcccctgtga 1140

taatgtcagc agctgtccct cctccgatac ctgctgccaa ctcacgtctg gggagtgggg 1200

ctgctgtcca atcccagagg ctgtctgctg ctcggaccac cagcactgct gcccccaggg 1260

ctacacgtgt gtagctgagg ggcagtgtca gcgaggaagc gagatcgtgg ctggactgga 1320

gaagatgcct gcccgccggg cttccttatc ccaccccaga gacatcggct gtgaccagca 1380

caccagctgc ccggtggggc agacctgctg cccgagcctg ggtggggagct gggcctgctg 1440

ccagttgccc catgctgtgt gctgcgagga tcgccagcac tgctgcccgg ctggctacac 1500

ctgcaacgtg aaggctcgat cctgcgagaa ggaagtggtc tctgcccagc ctgccacctt 1560

cctggcccgt agccctcacg tgggtgtgaa ggacgtggag tgtggggaag gacacttctg 1620

ccatgataac cagacctgct gccgagacaa ccgacagggc tgggcctgct gtccctaccg 1680

ccagggcgtc tgttgtgctg atcggcgcca ctgctgtcct gctggcttcc gctgcgcagc 1740

caggggtacc aagtgtttgc gcagggaggc cccgcgctgg gacgcccctt tgagggaccc 1800

agccttgaga cagctgctgt gagggacagt actgaagact ctgcagccct cgggacccca 1860

ctcggagggt gccctctgct caggcctccc tagcacctcc ccctaaccaa attctccctg 1920

gaccccattc tgagctcccc atcaccatgg gaggtggggc ctcaatctaa ggccttccct 1980

gtcagaaggg ggttgtggca aaagccacat tacaagctgc catcccctcc ccgtttcagt 2040

ggaccctgtg gccaggtgct tttccctatc cacaggggtg tttgtgtgtg tgcgcgtgtg 2100

cgtttcaata aagtttgtac actttcttaa 2130

<210> 10

<211> 171

<212> DNA

<213> Homo sapiens

<400> 10

gccatccagt gccccgacag ccagttcgag tgccccgact tcagcacctg ctgcgtgatg 60

gtggacggca gctggggctg ctgccccatg ccccaggcca gctgctgcga ggacagggtg 120

cactgctgcc cccacggcgc cttctgcgac ctggtgcaca ccaggtgcat c 171

<210> 11

<211> 168

<212> DNA

<213> Homo sapiens

<400> 11

gtgatgtgcc ccgacgccag gagcaggtgc cccgacggca gcacctgctg cgagctgccc 60

agcggcaagt acggctgctg ccccatgccc aacgccacct gctgcagcga ccacctgcac 120

tgctgccccc aggacaccgt gtgcgacctg atccagagca agtgcctg 168

<210> 12

<211> 162

<212> DNA

<213> Homo sapiens

<400> 12

gtgccctgcg acaacgtgag cagctgcccc agcagcgaca cctgctgcca gctgaccagc 60

ggcgagtggg gctgctgccc catccccgag gccgtgtgct gcagcgacca ccagcactgc 120

tgcccccagg gctacacctg cgtggccgag ggccagtgcc ag 162

<210> 13

<211> 168

<212> DNA

<213> Homo sapiens

<400> 13

gacgtggagt gcggcgaggg ccacttctgc cacgacaacc agacctgctg cagggacaac 60

aggcagggct gggcctgctg cccctacagg cagggcgtgt gctgcgccga caggaggcac 120

tgctgccccg ccggcttcag gtgcgccgcc aggggcacca agtgcctg 168

<210> 14

<211> 165

<212> DNA

<213> Homo sapiens

<400> 14

ggcccctgcc aggtggacgc ccactgcagc gccggccaca gctgcatctt caccgtgagc 60

ggcaccagca gctgctgccc cttccccgag gccgtggcct gcggcgacgg ccaccactgc 120

tgccccaggg gcttccactg cagcgccgac ggcaggagct gcttc 165

<210> 15

<211> 165

<212> DNA

<213> Homo sapiens

<400> 15

gtgaagtgcg acatggaggt gagctgcccc gacggctaca cctgctgcag gctgcagagc 60

ggcgcctggg gctgctgccc cttcacccag gccgtgtgct gcgaggacca catccactgc 120

tgccccgccg gcttcacctg cgacacccag aagggcacct gcgag 165

<210> 16

<211> 168

<212> DNA

<213> Homo sapiens

<400> 16

gacatcggct gcgaccagca caccagctgc cccgtgggcc agacctgctg ccccagcctg 60

ggcggcagct gggcctgctg ccagctgccc cacgccgtgt gctgcgagga caggcagcac 120

tgctgccccg ccggctacac ctgcaacgtg aaggccagga gctgcgag 168

<210> 17

<211> 252

<212> DNA

<213> Artificial sequence

<220>

<223> Signal sequence + GEM

<400> 17

atgtggaccc tggtgagctg ggtggccctg accgccggcc tggtggccgg ccagaggagc 60

ggcaacaaca gcgtgggcgc catccagtgc cccgacagcc agttcgagtg ccccgacttc 120

agcacctgct gcgtgatggt ggacggcagc tggggctgct gccccatgcc ccaggccagc 180

tgctgcgagg acagggtgca ctgctgcccc cacggcgcct tctgcgacct ggtgcacacc 240

aggtgcatct aa 252

<210> 18

<211> 300

<212> DNA

<213> Artificial sequence

<220>

<223> Signal sequence + GEM

<400> 18

atgtggaccc tggtgagctg ggtggccctg accgccggcc tggtggccgg cacccccacc 60

ggcacccacc ccctggccaa gaagctgccc gcccagagga ccaacagggc cgtggccctg 120

agcagcagcg tgatgtgccc cgacgccagg agcaggtgcc ccgacggcag cacctgctgc 180

gagctgccca gcggcaagta cggctgctgc cccatgccca acgccacctg ctgcagcgac 240

cacctgcact gctgccccca ggacaccgtg tgcgacctga tccagagcaa gtgcctgtaa 300

<210> 19

<211> 297

<212> DNA

<213> Artificial sequence

<220>

<223> Signal sequence + GEM

<400> 19

atgtggaccc tggtgagctg ggtggccctg accgccggcc tggtggccgg ccagggcccc 60

caccaggtgc cctggatgga gaaggccccc gcccacctga gcctgcccga cccccaggcc 120

ctgaagaggg acgtgccctg cgacaacgtg agcagctgcc ccagcagcga cacctgctgc 180

cagctgacca gcggcgagtg gggctgctgc cccatccccg aggccgtgtg ctgcagcgac 240

caccagcact gctgccccca gggctacacc tgcgtggccg agggccagtg ccagtaa 297

<210> 20

<211> 285

<212> DNA

<213> Artificial sequence

<220>

<223> Signal sequence + GEM

<400> 20

atgtggaccc tggtgagctg ggtggccctg accgccggcc tggtggccgg caaggaggtg 60

gtgagcgccc agcccgccac cttcctggcc aggagccccc acgtgggcgt gaaggacgtg 120

gagtgcggcg agggccactt ctgccacgac aaccagacct gctgcaggga caacaggcag 180

ggctgggcct gctgccccta caggcagggc gtgtgctgcg ccgacaggag gcactgctgc 240

cccgccggct tcaggtgcgc cgccaggggc accaagtgcc tgtaa 285

<210> 21

<211> 252

<212> DNA

<213> Artificial sequence

<220>

<223> Signal sequence + GEM

<400> 21

atgtggaccc tggtgagctg ggtggccctg accgccggcc tggtggccgg caagtggccc 60

accaccctga gcaggcacct gggcggcccc tgccaggtgg acgcccactg cagcgccggc 120

cacagctgca tcttcaccgt gagcggcacc agcagctgct gccccttccc cgaggccgtg 180

gcctgcggcg acggccacca ctgctgcccc aggggcttcc actgcagcgc cgacggcagg 240

agctgcttct aa 252

<210> 22

<211> 279

<212> DNA

<213> Artificial sequence

<220>

<223> Signal sequence + GEM

<400> 22

atgtggaccc tggtgagctg ggtggccctg accgccggcc tggtggccgg cagcaaggag 60

aacgccacca ccgacctgct gaccaagctg cccgcccaca ccgtgggcga cgtgaagtgc 120

gacatggagg tgagctgccc cgacggctac acctgctgca ggctgcagag cggcgcctgg 180

ggctgctgcc ccttcaccca ggccgtgtgc tgcgaggacc acatccactg ctgccccgcc 240

ggcttcacct gcgacaccca gaagggcacc tgcgagtaa 279

<210> 23

<211> 291

<212> DNA

<213> Artificial sequence

<220>

<223> Signal sequence + GEM

<400> 23

atgtggaccc tggtgagctg ggtggccctg accgccggcc tggtggccgg caggggcagc 60

gagatcgtgg ccggcctgga gaagatgccc gccaggaggg ccagcctgag ccaccccagg 120

gacatcggct gcgaccagca caccagctgc cccgtgggcc agacctgctg ccccagcctg 180

ggcggcagct gggcctgctg ccagctgccc cacgccgtgt gctgcgagga caggcagcac 240

tgctgccccg ccggctacac ctgcaacgtg aaggccagga gctgcgagta a 291

<210> 24

<211> 483

<212> DNA

<213> Artificial sequence

<220>

<223> Signal sequence + GEM

<400> 24

atgtggaccc tggtgagctg ggtggccctg accgccggcc tggtggccgg ccagaggagc 60

ggcaacaaca gcgtgggcgc catccagtgc cccgacagcc agttcgagtg ccccgacttc 120

agcacctgct gcgtgatggt ggacggcagc tggggctgct gccccatgcc ccaggccagc 180

tgctgcgagg acagggtgca ctgctgcccc cacggcgcct tctgcgacct ggtgcacacc 240

aggtgcatca aggaggtggt gagcgcccag cccgccacct tcctggccag gagcccccac 300

gtgggcgtga aggacgtgga gtgcggcgag ggccacttct gccacgacaa ccagacctgc 360

tgcagggaca acaggcaggg ctgggcctgc tgcccctaca ggcagggcgt gtgctgcgcc 420

gacaggaggc actgctgccc cgccggcttc aggtgcgccg ccaggggcac caagtgcctg 480

taa 483

<210> 25

<211> 963

<212> DNA

<213> Artificial sequence

<220>

<223> Signal sequence + GEM

<400> 25

atgtggaccc tggtgagctg ggtggccctg accgccggcc tggtggccgg ccagaggagc 60

ggcaacaaca gcgtgggcgc catccagtgc cccgacagcc agttcgagtg ccccgacttc 120

agcacctgct gcgtgatggt ggacggcagc tggggctgct gccccatgcc ccaggccagc 180

tgctgcgagg acagggtgca ctgctgcccc cacggcgcct tctgcgacct ggtgcacacc 240

aggtgcatcc agggccccca ccaggtgccc tggatggaga aggcccccgc ccacctgagc 300

ctgcccgacc cccaggccct gaagagggac gtgccctgcg acaacgtgag cagctgcccc 360

agcagcgaca cctgctgcca gctgaccagc ggcgagtggg gctgctgccc catccccgag 420

gccgtgtgct gcagcgacca ccagcactgc tgcccccagg gctacacctg cgtggccgag 480

ggccagtgcc agaggggcag cgagatcgtg gccggcctgg agaagatgcc cgccaggagg 540

gccagcctga gccaccccag ggacatcggc tgcgaccagc acaccagctg ccccgtgggc 600

cagacctgct gccccagcct gggcggcagc tgggcctgct gccagctgcc ccacgccgtg 660

tgctgcgagg acaggcagca ctgctgcccc gccggctaca cctgcaacgt gaaggccagg 720

agctgcgaga aggaggtggt gagcgcccag cccgccacct tcctggccag gagcccccac 780

gtgggcgtga aggacgtgga gtgcggcgag ggccacttct gccacgacaa ccagacctgc 840

tgcagggaca acaggcaggg ctgggcctgc tgcccctaca ggcagggcgt gtgctgcgcc 900

gacaggaggc actgctgccc cgccggcttc aggtgcgccg ccaggggcac caagtgcctg 960

taa 963

<210> 26

<211> 51

<212> DNA

<213> Homo sapiens

<400> 26

atgtggaccc tggtgagctg ggtggccctg accgccggcc tggtggccgg c 51

<210> 27

<211> 83

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence + linker/leader sequence + GEM F (GEM 1)

<400> 27

Met Trp Thr Leu Val Ser Trp Val Ala Leu Thr Ala Gly Leu Val Ala

1 5 10 15

Gly Gln Arg Ser Gly Asn Asn Ser Val Gly Ala Ile Gln Cys Pro Asp

20 25 30

Ser Gln Phe Glu Cys Pro Asp Phe Ser Thr Cys Cys Val Met Val Asp

35 40 45

Gly Ser Trp Gly Cys Cys Pro Met Pro Gln Ala Ser Cys Cys Glu Asp

50 55 60

Arg Val His Cys Cys Pro His Gly Ala Phe Cys Asp Leu Val His Thr

65 70 75 80

Arg Cys Ile

<210> 28

<211> 99

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence + linker + GEM

<400> 28

Met Trp Thr Leu Val Ser Trp Val Ala Leu Thr Ala Gly Leu Val Ala

1 5 10 15

Gly Thr Pro Thr Gly Thr His Pro Leu Ala Lys Lys Leu Pro Ala Gln

20 25 30

Arg Thr Asn Arg Ala Val Ala Leu Ser Ser Ser Val Met Cys Pro Asp

35 40 45

Ala Arg Ser Arg Cys Pro Asp Gly Ser Thr Cys Cys Glu Leu Pro Ser

50 55 60

Gly Lys Tyr Gly Cys Cys Pro Met Pro Asn Ala Thr Cys Cys Ser Asp

65 70 75 80

His Leu His Cys Cys Pro Gln Asp Thr Val Cys Asp Leu Ile Gln Ser

85 90 95

Lys Cys Leu

<210> 29

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence + linker + GEM

<400> 29

Met Trp Thr Leu Val Ser Trp Val Ala Leu Thr Ala Gly Leu Val Ala

1 5 10 15

Gly Gln Gly Pro His Gln Val Pro Trp Met Glu Lys Ala Pro Ala His

20 25 30

Leu Ser Leu Pro Asp Pro Gln Ala Leu Lys Arg Asp Val Pro Cys Asp

35 40 45

Asn Val Ser Ser Cys Pro Ser Ser Asp Thr Cys Cys Gln Leu Thr Ser

50 55 60

Gly Glu Trp Gly Cys Cys Pro Ile Pro Glu Ala Val Cys Cys Ser Asp

65 70 75 80

His Gln His Cys Cys Pro Gln Gly Tyr Thr Cys Val Ala Glu Gly Gln

85 90 95

CysGln

<210> 30

<211> 94

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence + linker + GEM

<400> 30

Met Trp Thr Leu Val Ser Trp Val Ala Leu Thr Ala Gly Leu Val Ala

1 5 10 15

Gly Lys Glu Val Val Ser Ala Gln Pro Ala Thr Phe Leu Ala Arg Ser

20 25 30

Pro His Val Gly Val Lys Asp Val Glu Cys Gly Glu Gly His Phe Cys

35 40 45

His Asp Asn Gln Thr Cys Cys Arg Asp Asn Arg Gln Gly Trp Ala Cys

50 55 60

Cys Pro Tyr Arg Gln Gly Val Cys Cys Ala Asp Arg Arg His Cys Cys

65 70 75 80

Pro Ala Gly Phe Arg Cys Ala Ala Arg Gly Thr Lys Cys Leu

85 90

<210> 31

<211> 83

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence + linker + GEM

<400> 31

Met Trp Thr Leu Val Ser Trp Val Ala Leu Thr Ala Gly Leu Val Ala

1 5 10 15

Gly Lys Trp Pro Thr Thr Leu Ser Arg His Leu Gly Gly Pro Cys Gln

20 25 30

Val Asp Ala His Cys Ser Ala Gly His Ser Cys Ile Phe Thr Val Ser

35 40 45

Gly Thr Ser Ser Cys Cys Pro Phe Pro Glu Ala Val Ala Cys Gly Asp

50 55 60

Gly His His Cys Cys Pro Arg Gly Phe His Cys Ser Ala Asp Gly Arg

65 70 75 80

Ser Cys Phe

<210> 32

<211> 92

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence + linker + GEM

<400> 32

Met Trp Thr Leu Val Ser Trp Val Ala Leu Thr Ala Gly Leu Val Ala

1 5 10 15

Gly Ser Lys Glu Asn Ala Thr Thr Asp Leu Leu Thr Lys Leu Pro Ala

20 25 30

His Thr Val Gly Asp Val Lys Cys Asp Met Glu Val Ser Cys Pro Asp

35 40 45

Gly Tyr Thr Cys Cys Arg Leu Gln Ser Gly Ala Trp Gly Cys Cys Pro

50 55 60

Phe Thr Gln Ala Val Cys Cys Glu Asp His Ile His Cys Cys Pro Ala

65 70 75 80

Gly Phe Thr Cys Asp Thr Gln Lys Gly Thr Cys Glu

85 90

<210> 33

<211> 96

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence + linker + GEM

<400> 33

Met Trp Thr Leu Val Ser Trp Val Ala Leu Thr Ala Gly Leu Val Ala

1 5 10 15

Gly Arg Gly Ser Glu Ile Val Ala Gly Leu Glu Lys Met Pro Ala Arg

20 25 30

Arg Ala Ser Leu Ser His Pro Arg Asp Ile Gly Cys Asp Gln His Thr

35 40 45

Ser Cys Pro Val Gly Gln Thr Cys Cys Pro Ser Leu Gly Gly Ser Trp

50 55 60

Ala Cys Cys Gln Leu Pro His Ala Val Cys Cys Glu Asp Arg Gln His

65 70 75 80

Cys Cys Pro Ala Gly Tyr Thr Cys Asn Val Lys Ala Arg Ser Cys Glu

85 90 95

<210> 34

<211> 160

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence + linker + GEM

<400> 34

Met Trp Thr Leu Val Ser Trp Val Ala Leu Thr Ala Gly Leu Val Ala

1 5 10 15

Gly Gln Arg Ser Gly Asn Asn Ser Val Gly Ala Ile Gln Cys Pro Asp

20 25 30

Ser Gln Phe Glu Cys Pro Asp Phe Ser Thr Cys Cys Val Met Val Asp

35 40 45

Gly Ser Trp Gly Cys Cys Pro Met Pro Gln Ala Ser Cys Cys Glu Asp

50 55 60

Arg Val His Cys Cys Pro His Gly Ala Phe Cys Asp Leu Val His Thr

65 70 75 80

Arg Cys Ile Lys Glu Val Val Ser Ala Gln Pro Ala Thr Phe Leu Ala

85 90 95

Arg Ser Pro His Val Gly Val Lys Asp Val Glu Cys Gly Glu Gly His

100 105 110

Phe Cys His Asp Asn Gln Thr Cys Cys Arg Asp Asn Arg Gln Gly Trp

115 120 125

Ala Cys Cys Pro Tyr Arg Gln Gly Val Cys Cys Ala Asp Arg Arg His

130 135 140

Cys Cys Pro Ala Gly Phe Arg Cys Ala Ala Arg Gly Thr Lys Cys Leu

145 150 155 160

<210> 35

<211> 320

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence + linker + GEM

<400> 35

Met Trp Thr Leu Val Ser Trp Val Ala Leu Thr Ala Gly Leu Val Ala

1 5 10 15

Gly Gln Arg Ser Gly Asn Asn Ser Val Gly Ala Ile Gln Cys Pro Asp

20 25 30

Ser Gln Phe Glu Cys Pro Asp Phe Ser Thr Cys Cys Val Met Val Asp

35 40 45

Gly Ser Trp Gly Cys Cys Pro Met Pro Gln Ala Ser Cys Cys Glu Asp

50 55 60

Arg Val His Cys Cys Pro His Gly Ala Phe Cys Asp Leu Val His Thr

65 70 75 80

Arg Cys Ile Gln Gly Pro His Gln Val Pro Trp Met Glu Lys Ala Pro

85 90 95

Ala His Leu Ser Leu Pro Asp Pro Gln Ala Leu Lys Arg Asp Val Pro

100 105 110

Cys Asp Asn Val Ser Ser Cys Pro Ser Ser Asp Thr Cys Cys Gln Leu

115 120 125

Thr Ser Gly Glu Trp Gly Cys Cys Pro Ile Pro Glu Ala Val Cys Cys

130 135 140

Ser Asp His Gln His Cys Cys Pro Gln Gly Tyr Thr Cys Val Ala Glu

145 150 155 160

Gly Gln Cys Gln Arg Gly Ser Glu Ile Val Ala Gly Leu Glu Lys Met

165 170 175

Pro Ala Arg Arg Ala Ser Leu Ser His Pro Arg Asp Ile Gly Cys Asp

180 185 190

Gln His Thr Ser Cys Pro Val Gly Gln Thr Cys Cys Pro Ser Leu Gly

195 200 205

Gly Ser Trp Ala Cys Cys Gln Leu Pro His Ala Val Cys Cys Glu Asp

210 215 220

Arg Gln His Cys Cys Pro Ala Gly Tyr Thr Cys Asn Val Lys Ala Arg

225 230 235 240

Ser Cys Glu Lys Glu Val Val Ser Ala Gln Pro Ala Thr Phe Leu Ala

245 250 255

Arg Ser Pro His Val Gly Val Lys Asp Val Glu Cys Gly Glu Gly His

260 265 270

Phe Cys His Asp Asn Gln Thr Cys Cys Arg Asp Asn Arg Gln Gly Trp

275 280 285

Ala Cys Cys Pro Tyr Arg Gln Gly Val Cys Cys Ala Asp Arg Arg His

290 295 300

Cys Cys Pro Ala Gly Phe Arg Cys Ala Ala Arg Gly Thr Lys Cys Leu

305 310 315 320

<210> 36

<211> 71

<212> PRT

<213> Artificial sequence

<220>

<223> GEM

<400> 36

Ala Met Asp Ile Gly Cys Asp Gln His Thr Ser Cys Pro Val Gly Gln

1 5 10 15

Thr Cys Cys Pro Ser Leu Gly Gly Ser Trp Ala Cys Cys Gln Leu Pro

20 25 30

His Ala Val Cys Cys Glu Asp Arg Gln His Cys Cys Pro Ala Gly Tyr

35 40 45

Thr Cys Asn Val Lys Ala Arg Ser Cys Glu Lys Leu Ala Ala Ala Leu

50 55 60

Glu His His His His His His

65 70

<210> 37

<211> 72

<212> PRT

<213> Artificial sequence

<220>

<223> GEM

<400> 37

Ala Met Ala Ile Gln Cys Pro Asp Ser Gln Phe Glu Cys Pro Asp Phe

1 5 10 15

Ser Thr Cys Cys Val Met Val Asp Gly Ser Trp Gly Cys Cys Pro Met

20 25 30

Pro Gln Ala Ser Cys Cys Glu Asp Arg Val His Cys Cys Pro His Gly

35 40 45

Ala Phe Cys Asp Leu Val His Thr Arg Cys Ile Lys Leu Ala Ala Ala

50 55 60

Leu Glu His His His His His

65 70

<210> 38

<211> 6389

<212> DNA

<213> Artificial sequence

<220>

<223> Carrier

<400> 38

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc ttctagacaa ctttgtatag aaaagttgcg ttacataact 180

tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaatagt 240

aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 300

cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg 360

taaatggccc gcctggcatt gtgcccagta catgacctta tgggactttc ctacttggca 420

gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt 480

cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt 540

attttgtgca gcgatggggg cgggggggggg gggggggcgc gcgccaggcg gggcggggcg 600

gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc 660

gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag 720

cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc 780

ctcgcgccgc ccgccccggc tctgactgac cgcgttatactc ccacaggtga gcgggcggga 840

cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta agggatggtt 900

ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca ctttttttca 960

ggttggcaag tttgtacaaa aaagcaggct gccaccatgt ggaccctggt gagctgggtg 1020

gccttaacag cagggctggt ggctggaacg cggtgcccag atggtcagtt ctgccctgtg 1080

gcctgctgcc tggaccccgg aggagccagc tacagctgct gccgtcccct tctggacaaa 1140

tggcccacaa cactgagcag gcatctgggt ggcccctgcc aggttgatgc ccactgctct 1200

gccggccact cctgcatctt taccgtctca gggacttcca gttgctgccc cttcccagag 1260

gccgtggcat gcggggatgg ccatcactgc tgcccacggg gcttccactg cagtgcagac 1320

gggcgatcct gcttccaaag atcaggtaac aactccgtgg gtgccatcca gtgccctgat 1380

agtcagttcg aatgccccgga cttctccacg tgctgtgtta tggtcgatgg ctcctggggg 1440

tgctgcccca tgccccaggc ttcctgctgt gaagacaggg tgcactgctg tccgcacggt 1500

gccttctgcg acctggttca cacccgctgc atcacaccca cgggcaccca ccccctggca 1560

aagaagctcc ctgcccagag gactaacagg gcagtggcct tgtccagctc ggtcatgtgt 1620

ccggacgcac ggtcccggtg ccctgatggt tctacctgct gtgagctgcc cagtgggaag 1680

tatggctgct gcccaatgcc caacgccacc tgctgctccg atcacctgca ctgctgcccc 1740

caagacactg tgtgtgacct gatccagagt aagtgcctct ccaaggagaa cgctaccacg 1800

gacctcctca ctaagctgcc tgcgcacaca gtgggggatg tgaaatgtga catggaggtg 1860

agctgcccag atggctatac ctgctgccgt ctacagtcgg gggcctgggg ctgctgccct 1920

tttacccagg ctgtgtgctg tgaggaccac atacactgct gtcccgcggg gtttacgtgt 1980

gacacgcaga agggtacctg tgaacagggg ccccaccagg tgccctggat ggagaaggcc 2040

ccagctcacc tcagcctgcc agacccacaa gccttgaaga gagatgtccc ctgtgataat 2100

gtcagcagct gtccctcctc cgatacctgc tgccaactca cgtctgggga gtggggctgc 2160

tgtccaatcc cagaggctgt ctgctgctcg gaccaccagc actgctgccc ccagggctac 2220

acgtgtgtag ctgaggggca gtgtcagcga ggaagcgaga tcgtggctgg actggagaag 2280

atgcctgccc gccgggcttc cttatcccac cccagagaca tcggctgtga ccagcacacc 2340

agctgcccgg tggggcagac ctgctgcccg agcctgggtg ggagctgggc ctgctgccag 2400

ttgccccatg ctgtgtgctg cgaggatcgc cagcactgct gcccggctgg ctacacctgc 2460

aacgtgaagg ctcgatcctg cgagaaggaa gtggtctctg cccagcctgc caccttcctg 2520

gcccgtagcc ctcacgtggg tgtgaaggac gtggagtgtg gggaaggaca cttctgccat 2580

gataaccaga cctgctgccg agacaaccga cagggctggg cctgctgtcc ctaccgccag 2640

ggcgtctgtt gtgctgatcg gcgccactgc tgtcctgctg gcttccgctg cgcagccagg 2700

ggtaccaagt gtttgcgcag ggaggccccg cgctgggacg cccctttgag ggacccagcc 2760

ttgagacagc tgctgtgaac ccagctttct tgtacaaagt gggaattccg ataatcaacc 2820

tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 2880

gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 2940

cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 3000

tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 3060

cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 3120

ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 3180

tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt 3240

tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 3300

ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg 3360

ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgggaat tcctagagct 3420

cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 3480

gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 3540

attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 3600

agcaaggggg aggattggga agagaatagc aggcatgctg gggagggccg caggaacccc 3660

tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac 3720

caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca 3780

gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 3840

accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg 3900

ggtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt 3960

cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 4020

ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 4080

tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 4140

gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc 4200

tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa 4260

aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat 4320

tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca 4380

cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 4440

acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 4500

acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 4560

aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 4620

tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 4680

gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 4740

tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 4800

aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 4860

cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 4920

agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 4980

ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 5040

tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 5100

tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 5160

caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 5220

accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 5280

attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 5340

ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 5400

taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg 5460

taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 5520

aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 5580

agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 5640

ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 5700

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 5760

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 5820

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 5880

tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 5940

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 6000

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 6060

gggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 6120

acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 6180

ggtaagcggc agggtcggaa caggagagcg cacgaggggag cttccagggg gaaacgcctg 6240

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 6300

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 6360

ggccttttgc tggccttttg ctcacatgt 6389

<210> 39

<211> 6932

<212> DNA

<213> Artificial sequence

<220>

<223> Carrier

<400> 39

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc ttctagacaa ctttgtatag aaaagttgct cgacattgat 180

tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg 240

agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 300

gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt 360

gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 420

atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 480

cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 540

ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct 600

ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg 660

ggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga 720

ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg 780

cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg 840

cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct 900

gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta 960

attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa agccttgagg 1020

ggctccggga gggccctttg tgcgggggga gcggctcggg gggtgcgtgc gtgtgtgtgt 1080

gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc tgcgggcgcg 1140

gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg ggcggtgccc 1200

cgcggtgcgg gggggctgc gaggggaaca aaggctgcgt gcggggtgtg tgcgtggggg 1260

ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac ccccctcccc 1320

gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt ggcgcggggc 1380

tcgccgtgcc gggcgggggg tggcggcagg tggggggtgcc gggcggggcg gggccgcctc 1440

gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc ggctgtcgag 1500

gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg cagggacttc 1560

ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc ccctctagcg 1620

ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag ggccttcgtg 1680

cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg cggggggacg 1740

gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct 1800

ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg 1860

tgctggttat tgtgctgtct catcattttg gcaaagaatt gcaagtttgt acaaaaaagc 1920

aggctgccac catgtggacc ctggtgagct gggtggcctt aacagcaggg ctggtggctg 1980

gaacgcggtg cccagatggt cagttctgcc ctgtggcctg ctgcctggac cccggaggag 2040

ccagctacag ctgctgccgt ccccttctgg acaaatggcc cacaacactg agcaggcatc 2100

tgggtggccc ctgccaggtt gatgcccact gctctgccgg ccactcctgc atctttaccg 2160

tctcagggac ttccagttgc tgccccttcc cagaggccgt ggcatgcggg gatggccatc 2220

actgctgccc acggggcttc cactgcagtg cagacgggcg atcctgcttc caaagatcag 2280

gtaacaactc cgtgggtgcc atccagtgcc ctgatagtca gttcgaatgc ccggacttct 2340

ccacgtgctg tgttatggtc gatggctcct gggggtgctg ccccatgccc caggcttcct 2400

gctgtgaagacagggtgcac tgctgtccgc acggtgcctt ctgcgacctg gttcacaccc 2460

gctgcatcac acccacgggc acccacccccc tggcaaagaa gctccctgcc cagaggacta 2520

acagggcagt ggccttgtcc agctcggtca tgtgtccgga cgcacggtcc cggtgccctg 2580

atggttctac ctgctgtgag ctgcccagtg ggaagtatgg ctgctgccca atgcccaacg 2640

ccacctgctg ctccgatcac ctgcactgct gcccccaaga cactgtgtgt gacctgatcc 2700

agagtaagtg cctctccaag gagaacgcta ccacggacct cctcactaag ctgcctgcgc 2760

acacagtggg ggatgtgaaa tgtgacatgg aggtgagctg cccagatggc tatacctgct 2820

gccgtctaca gtcgggggcc tggggctgct gcccttttac ccaggctgtg tgctgtgagg 2880

accacataca ctgctgtccc gcggggttta cgtgtgacac gcagaagggt acctgtgaac 2940

aggggcccca ccaggtgccc tggatggaga aggccccagc tcacctcagc ctgccagacc 3000

cacaagcctt gaagagagat gtcccctgtg ataatgtcag cagctgtccc tcctccgata 3060

cctgctgcca actcacgtct gggagtggg gctgctgtcc aatcccagag gctgtctgct 3120

gctcggacca ccagcactgc tgcccccagg gctacacgtg tgtagctgag gggcagtgtc 3180

agcgaggaag cgagatcgtg gctggactgg agaagatgcc tgcccgccgg gcttccttat 3240

cccaccccag agacatcggc tgtgaccagc acaccagctg cccggtgggg cagacctgct 3300

gcccgagcct gggtgggagc tgggcctgct gccagttgcc ccatgctgtg tgctgcgagg 3360

atcgccagca ctgctgcccg gctggctaca cctgcaacgt gaaggctcga tcctgcgaga 3420

aggaagtggt ctctgcccag cctgccacct tcctggcccg tagccctcac gtgggtgtga 3480

aggacgtgga gtgtggggaa ggacacttct gccatgataa ccagacctgc tgccgagaca 3540

accgacaggg ctgggcctgc tgtccctacc gccagggcgt ctgttgtgct gatcggcgcc 3600

actgctgtcc tgctggcttc cgctgcgcag ccaggggtac caagtgtttg cgcagggagg 3660

ccccgcgctg ggacgcccct ttgagggacc cagccttgag acagctgctg tgaatcgaat 3720

tctcgagata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac 3780

tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt 3840

gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttagt tcttgccacg 3900

gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact 3960

gacaattccg tggtgaccca gctttcttgt acaaagtggg aattcctaga gctcgctgat 4020

cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 4080

ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 4140

cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 4200

gggaggattg ggaagagaat agcaggcatg ctggggaggg ccgcaggaac ccctagtgat 4260

ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt 4320

cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct 4380

gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat 4440

acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg 4500

gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc 4560

ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc 4620

cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt 4680

gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag 4740

tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg 4800

ggctattcttttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag 4860

ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg 4920

tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca 4980

acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct 5040

gtgaccgtct ccggggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg 5100

agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt 5160

tcttagacgt cctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat 5220

atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg 5280

agccatattc aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta 5340

tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg 5400

tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat 5460

gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc 5520

atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga 5580

aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg 5640

ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc 5700

gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg 5760

agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat 5820

aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac 5880

cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca 5940

gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta 6000

cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt 6060

catttgatgc tcgatgagtt tttctatcag aattggttaa ttggttgtaa cactggcaga 6120

gcattacgct gacttgacgg gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt 6180

acgcgtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 6240

ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 6300

gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 6360

ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 6420

aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 6480

gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 6540

gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 6600

aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 6660

cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 6720

tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 6780

ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 6840

atgctcgtca gggggcgga gcctatggaa aaacgccagc aacgcggcctttttacggtt 6900

cctggccttt tgctggcctt ttgctcacat gt 6932

Claims (29)

1. A recombinant polypeptide or a combination of recombinant polypeptides comprising two to six granulin/epicopin modules (GEMs).

2. The recombinant polypeptide or combination of claim 1, comprising two to six granulin/epicopin modules (GEM), including GEM E (4), and one or more of additional GEM F (1), GEM B (2), GEM C (3), and GEM D (7).

3. The recombinant polypeptide or combination of claim 1, comprising a granulin/epigenin module (GEM) E, and an additional GEM F.

4. The recombinant polypeptide or combination of claim 1, comprising a granulin/epigenin module (GEM) E, and an additional GEM C.

5. The recombinant polypeptide or combination of claim 1, comprising a granulin/epigenin module (GEM) E, and an additional GEM B.

6. The recombinant polypeptide or combination of claim 1, comprising a granulin/epigenin module (GEM) E, and additional GEM F and GEM D.

7. The recombinant polypeptide or combination of claim 1, comprising a granulin/epicopexin module (GEM) E, and additional GEM F, GEM D, and GEM C.

8. The recombinant polypeptide or combination of claim 1, comprising two to six granulin/epicopexin modules (GEM), including GEM F, and one or more of additional GEM a, GEM B, GEM E, GEM C, and GEM D.

9. The recombinant polypeptide or combination of claim 1, comprising a granulin/epigenin module (GEM) F, and an additional GEM B.

10. The recombinant polypeptide or combination of claim 1, comprising a granulin/epigenin module (GEM) F, and an additional GEM a.

11. The recombinant polypeptide or combination of claim 1, comprising a granulin/epigenin module (GEM) F, and an additional GEM D.

12. The recombinant polypeptide or combination of claim 1, comprising a signal sequence at the N-terminus of the GEM.

13. The recombinant polypeptide or combination of claim 1, comprising one or more linker (leader) sequences located at the N-terminus and/or in between the GEMs.

14. The recombinant polypeptide or combination according to claim 1, wherein the polypeptide comprises or consists of a mixed portion of full length PGRN sequences according to SEQ ID NO 1 and/or does not consist of full length PGRN sequences according to SEQ ID NO 1.

15. The recombinant polypeptide or combination of claim 1, wherein:

gem E comprises or consists of SEQ ID NO 5,

gem F comprises or consists of SEQ ID NO 2,

gem C comprises or consists of SEQ ID NO 4,

gem D comprises or consists of SEQ ID NO 8,

gem a comprises or consists of SEQ ID NO 7, and/or

Gem B comprises or consists of SEQ ID NO 3.

16. The recombinant polypeptide or combination according to claim 12, wherein the signal sequence comprises or consists of SEQ ID NO 26.

17. The recombinant polypeptide or combination of claim 13, wherein the linker sequence comprises or consists of: the sequence according to SEQ ID NO 27-35 or one or more linker sequences within the sequence according to SEQ ID NO 27-35.

18. The recombinant polypeptide or combination of recombinant polypeptides according to claim 1, comprising or consisting of one or more of SEQ ID NOs 27-35.

19. A combination of multiple recombinant polypeptides, the combination comprising two to six granulin/epicoppered modules (GEM) according to claim 1, comprising GEM E (4) and one or more of additional GEM B (2), GEM F (1), GEM C (3) and GEM D (7), and/or comprising two to six granulin/epicoppered modules (GEM) comprising GEM F and one or more of additional GEM B, GEM E, GEM a and GEM D.

20. A nucleic acid molecule encoding the recombinant polypeptide or combination of recombinant polypeptides according to claim 1.

21. The nucleic acid molecule of claim 20, which is present as a combination of a plurality of nucleic acid molecules, each encoding one or more of the recombinant polypeptides of claim 1.

22. The nucleic acid molecule of claim 20 or 21, in the form of a vector configured to express the recombinant polypeptide of claim 1 upon administration to a subject.

23. The nucleic acid molecule of claim 20 or 21, wherein the vector is selected from the group consisting of adenovirus, adeno-associated virus, lentivirus, and baculovirus.

24. The nucleic acid molecule of claim 20 or 21, wherein the molecule encodes a plurality of GEMs configured for expression as polycistronic mRNA, wherein the GEMs are encoded by a single nucleic acid molecule and configured for post-transcriptional and/or post-translational cleavage, and/or wherein the polycistronic mRNA comprises a plurality of Internal Ribosome Entry Sites (IRES) enabling expression of a plurality of different and soluble GEM polypeptides.

25. The nucleic acid molecule of claim 20 or 21, wherein the molecule encodes a plurality of GEMs configured to be expressed under the control of a plurality of promoters such that a plurality of different and soluble GEM polypeptides can be expressed.

26. A pharmaceutical composition comprising a recombinant polypeptide or combination according to claim 1, a combination of a plurality of recombinant polypeptides according to claim 18, or a nucleic acid molecule according to claim 20 or 21, together with a pharmaceutically acceptable excipient.

27. A method of treating a neurodegenerative disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide or combination of claim 1, a combination of a plurality of recombinant polypeptides of claim 18, a nucleic acid molecule of claim 20 or 21, or a composition of claim 26.

28. The method of claim 27, wherein the neurodegenerative disease is selected from the group consisting of: amyotrophic Lateral Sclerosis (ALS), frontotemporal dementia (FTD), spinal Muscular Atrophy (SMA), alzheimer's Disease (AD), and Parkinson's Disease (PD).

29. The method of claim 27, wherein the neurodegenerative disease is selected from diseases associated with aberrant lysosomal function, such as Parkinson's Disease (PD), gaucher's disease, or Neuronal Ceroid Lipofuscinosis (NCL).