WO2021081201A1 - Adeno-associated virus (aav) systems for treatment of progranulin associated neurodegeneative diseases or disorders - Google Patents

Adeno-associated virus (aav) systems for treatment of progranulin associated neurodegeneative diseases or disorders Download PDF

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WO2021081201A1
WO2021081201A1 PCT/US2020/056860 US2020056860W WO2021081201A1 WO 2021081201 A1 WO2021081201 A1 WO 2021081201A1 US 2020056860 W US2020056860 W US 2020056860W WO 2021081201 A1 WO2021081201 A1 WO 2021081201A1
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Prior art keywords
aav
nucleic acid
seq
polynucleotide
promoter
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French (fr)
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WO2021081201A8 (en
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Juan Li
Tanaz FARIVAR
Savitri MANDAPADI
Steven Pennock
Mark Shearman
Judith NEWMARK
Adrian Timmers
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Applied Genetic Technologies Corp
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Applied Genetic Technologies Corp
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Priority to AU2020371662A priority Critical patent/AU2020371662A1/en
Priority to KR1020227017064A priority patent/KR20230019402A/ko
Priority to CA3158516A priority patent/CA3158516A1/en
Priority to IL292382A priority patent/IL292382A/en
Priority to EP20880235.5A priority patent/EP4048799A4/en
Priority to CN202080089386.1A priority patent/CN115715327A/zh
Priority to JP2022523408A priority patent/JP2022553307A/ja
Priority to MX2022004812A priority patent/MX2022004812A/es
Publication of WO2021081201A1 publication Critical patent/WO2021081201A1/en
Publication of WO2021081201A8 publication Critical patent/WO2021081201A8/en
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C12N2710/16011—Herpesviridae
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    • C12N2750/14011—Parvoviridae
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    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • the present invention relates to the field of gene therapy, including AAV vectors for expressing an isolated polynucleotides in a subject or cell.
  • the disclosure also relates to nucleic acid constructs, promoters, vectors, and host cells including the polynucleotides as well as methods of delivering exogenous DNA sequences to a target cell, tissue, organ or organism, and methods for use in the treatment or prevention of progranulin associated neurodegenerative diseases or disorders.
  • Gene therapy aims to improve clinical outcomes for patients suffering from either genetic mutations or acquired diseases caused by an aberration in the gene expression profile.
  • Gene therapy includes the treatment or prevention of medical conditions resulting from defective genes or abnormal regulation or expression, e.g. underexpression or overexpression, that can result in a disorder, disease, malignancy, etc.
  • a disease or disorder caused by a defective gene might be treated, prevented or ameliorated by delivery of a corrective genetic material to a patient, or might be treated, prevented or ameliorated by altering or silencing a defective gene, e.g., with a corrective genetic material to a patient resulting in the therapeutic expression of the genetic material within the patient.
  • the basis of gene therapy is to supply a transcription cassette with an active gene product (sometimes referred to as a transgene or a therapeutic nucleic acid), e.g., that can result in a positive gain- of-function effect, a negative loss-of-fimction effect, or another outcome.
  • an active gene product sometimes referred to as a transgene or a therapeutic nucleic acid
  • Such outcomes can be attributed to expression of a therapeutic protein such as an antibody, a functional enzyme, or a fusion protein.
  • Gene therapy can also be used to treat a disease or malignancy caused by other factors. Human monogenic disorders can be treated by the delivery and expression of a normal gene to the target cells. Delivery and expression of a corrective gene in the patient's target cells can be carried out via numerous methods, including the use of engineered viruses and viral gene delivery vectors.
  • Adeno-associated viruses belong to the Parvoviridae family and more specifically constitute the dependoparvovirus genus.
  • Vectors derived from AAV i.e., recombinant AAV (rAVV) or AAV vectors
  • rAVV recombinant AAV
  • 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 are devoid of the virus structural genes, thereby diminishing the host cell responses to virus infection, e.g., interferon-mediated responses;
  • wild-type viruses are considered non-pathologic in humans;
  • replication- deficient AAV vectors lack the rep gene and generally persist as episomes, thus limiting the risk of insertional mutagenesis or genotoxicity; and (v) in comparison to other vector systems, AAV vectors are generally considered to be relatively poor immunogen
  • Progranulin is a widely expressed, secreted glycoprotein that acts as a trophic factor for many cell types, including neuronal cells, modulates inflammation, and facilitates wound repair.
  • PGRN is involved in the regulation of multiple processes including development, wound healing, angiogenesis, growth and maintenance of neuronal cells, and inflammation.
  • PGRN is expressed in neurons and microglia (Petkau et al, Neurol. 2010. 518, 3931-3947) and has been implicated in inflammation (Yin el al. , J. Exp. Med. 2010. 207, 117-12; Tang et al. , Science. 2010; 332, 478-484), wound repair (He et al. , Nat. Med . 2003.
  • Sortilin significantly increases plasma PGRN levels both in mouse models in vivo and human cells In vitro (Carrasquillo. M. M et al, 2010. Am J Hum Genet 87, 890-897; Lee, W. C et al, 2010. 23, 1467- 1478).
  • a polymorphism in Sortilin was shown to be strongly associated with PGRN serum levies in humans (Carrasquillo M. et al, 2010. Am J Hum Genet. 10; 87(6):890-7). Tanaka et al, Hum Mol Genet. 2017 Mar l;26(5):969-988; Paushter et al, Acta Neuropathol.
  • PGRN expression has been shown in multiple neurodegenerative disorders, and recent studies into the genetic etiology of neurodegenerative diseases have shown that heritable mutations in the PGRN gene may lead to adult-onset neurodegenerative disorders due to reduced neuronal survival.
  • Complete PGRN deficiency i.e., homozygous PGRN mutants
  • loss-of-function mutations lead to the neurodegenerative diseases or disorders, including but not limited to familial frontotemporal dementia (FTD), and the neurodegenerative lysosomal storage disorder neuronal ceroid lipofuscinosis (NCL), including neuronal ceroid lipofuscinosis 11 (CLN11).
  • Frontotemporal dementia encompasses a group of neurodegenerative disorders characterized by cognitive and behavioral impairments.
  • Heterozygous mutations in progranulin (PGRN) cause familial FTD and result in decreased PGRN expression, while homozygous mutations result in complete loss of PGRN expression and lead to NCL.
  • CLN11 also termed adult-onset CLN, shares some clinical features with FTD such as cognitive decline and eventual fatality, but is also characterized by progressive visual loss via retinal dystrophy, seizures, cerebellar ataxia, and cerebellar atrophy.
  • Low PGRN promotes neuroinflammation and enhances peripheral inflammatory conditions such as arthritis and atherosclerosis, and thus any disorder characterized by neuroinflammation or peripheral inflammation could be a potential target for PGRN.
  • low PGRN is risk factor for schizophrenia, bipolar and psychiatric disorders (Chitramuthu et al, Brain. 2017 Dec 1 ;140(12):3081-3104).
  • FTD is a fatal, degenerative brain disease that is a common cause of dementia in people under the age of 60. Often striking people in their prime, FTD is characterized by a progressive degeneration of the frontal portions of the brain, the regions responsible for language and behavior. Over the course of the disease, FTD patients may lose the ability to behave appropriately, make judgements, communicate, and carry out daily activities.
  • Frontotemporal lobar degeneration (FTLD) refers to a collection of pathologic diagnoses that can cause FTD syndromes.
  • Progranulin supplementation may be used to treat multiple disease conditions including neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and Parkinson’s-like diseases (Capell et al, 2011; Cenik et al, 2011; Van Kampen et al, 2014; Minami et al, 2015), acute brain injury (Tao et al, 2012; Egashira et al, 2013; Jackman et al, 2013; Kanazawa et al, 2015; Zhao and Bateman, 2015; Altmann et al, 2016b; Xie et al, 2016). Furthermore, progranulin has been suggested as a therapeutic target for many peripheral conditions particularly those with an important inflammatory component (He et al, 2003; Sfikakis and Tsokos,
  • the disclosure relates to recombinant adeno-associated virus (rAAV) vectors through which a progranulin expression cassette can be packaged for CNS-targeted delivery to patients suffering from neurodegenerative disorders associated with progranulin (PGRN) mutations.
  • rAAV adeno-associated virus
  • the disclosure provides an isolated polynucleotide comprising a nucleic acid sequence encoding progranulin.
  • the nucleic acid sequence is a non-naturally occurring sequence.
  • the nucleic acid sequence encodes mammalian progranulin.
  • the mammalian progranulin is human progranulin.
  • the nucleic acid comprises a sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.
  • the nucleic acid comprises a sequence that is at least 85% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.
  • the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 5.
  • the nucleic acid comprises a sequence that is at least 90% identical to SEQ ID NO: 5.
  • the nucleic acid comprises a sequence that is at least 95% identical to SEQ ID NO: 5.
  • the nucleic acid comprises a sequence that is at least 96% identical to SEQ ID NO: 5. According to some embodiments, the nucleic acid comprises a sequence that is at least 97% identical to SEQ ID NO: 5. According to some embodiments, the nucleic acid comprises a sequence that is at least 98% identical to SEQ ID NO: 5. According to some embodiments, the nucleic acid comprises a sequence that is at least 99% identical to SEQ ID NO: 5. According to some embodiments, the nucleic acid consists of SEQ ID NO: 5. According to some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 6.
  • the nucleic acid comprises a sequence that is at least 90% identical to SEQ ID NO: 6. According to some embodiments, the nucleic acid comprises a sequence that is at least 95% identical to SEQ ID NO: 6. According to some embodiments, the nucleic acid comprises a sequence that is at least 96% identical to SEQ ID NO: 6. According to some embodiments, the nucleic acid comprises a sequence that is at least 97% identical to SEQ ID NO: 6. According to some embodiments, the nucleic acid comprises a sequence that is at least 98% identical to SEQ ID NO: 6. According to some embodiments, the nucleic acid comprises a sequence that is at least 99% identical to SEQ ID NO: 6.
  • the nucleic acid consists of SEQ ID NO: 6. According to some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 7. According to some embodiments, the nucleic acid comprises a sequence that is at least 90% identical to SEQ ID NO: 7. According to some embodiments, the nucleic acid comprises a sequence that is at least 95% identical to SEQ ID NO: 7. According to some embodiments, the nucleic acid comprises a sequence that is at least 96% identical to SEQ ID NO: 7. According to some embodiments, the nucleic acid comprises a sequence that is at least 97% identical to SEQ ID NO: 7.
  • the nucleic acid comprises a sequence that is at least 98% identical to SEQ ID NO: 7. According to some embodiments, the nucleic acid comprises a sequence that is at least 99% identical to SEQ ID NO: 7. According to some embodiments, the nucleic acid consists of SEQ ID NO: 7. According to some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 8. According to some embodiments, the nucleic acid comprises a sequence that is at least 90% identical to SEQ ID NO: 8. According to some embodiments, the nucleic acid comprises a sequence that is at least 95% identical to SEQ ID NO: 8.
  • the nucleic acid comprises a sequence that is at least 96% identical to SEQ ID NO: 8. According to some embodiments, the nucleic acid comprises a sequence that is at least 97% identical to SEQ ID NO: 8. According to some embodiments, the nucleic acid comprises a sequence that is at least 98% identical to SEQ ID NO: 8. According to some embodiments, the nucleic acid comprises a sequence that is at least 99% identical to SEQ ID NO: 8. According to some embodiments, the nucleic acid consists of SEQ ID NO: 8. According to some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 9.
  • the nucleic acid comprises a sequence that is at least 90% identical to SEQ ID NO: 9. According to some embodiments, the nucleic acid comprises a sequence that is at least 95% identical to SEQ ID NO: 9. According to some embodiments, the nucleic acid comprises a sequence that is at least 96% identical to SEQ ID NO: 9. According to some embodiments, the nucleic acid comprises a sequence that is at least 97% identical to SEQ ID NO: 9. According to some embodiments, the nucleic acid comprises a sequence that is at least 98% identical to SEQ ID NO: 9. According to some embodiments, the nucleic acid comprises a sequence that is at least 99% identical to SEQ ID NO: 9.
  • the nucleic acid consists of SEQ ID NO: 9. According to some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 10. According to some embodiments, the nucleic acid comprises a sequence that is at least 90% identical to SEQ ID NO: 10. According to some embodiments, the nucleic acid comprises a sequence that is at least 95% identical to SEQ ID NO: 10. According to some embodiments, the nucleic acid comprises a sequence that is at least 96% identical to SEQ ID NO: 10. According to some embodiments, the nucleic acid comprises a sequence that is at least 97% identical to SEQ ID NO:
  • the nucleic acid comprises a sequence that is at least 98% identical to SEQ ID NO: 10. According to some embodiments, the nucleic acid comprises a sequence that is at least 99% identical to SEQ ID NO: 10. According to some embodiments, the nucleic acid consists of SEQ ID NO: 10. According to some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 11. According to some embodiments, the nucleic acid comprises a sequence that is at least 90% identical to SEQ ID NO: 11. According to some embodiments, the nucleic acid comprises a sequence that is at least 95% identical to SEQ ID NO: 11. According to some embodiments, the nucleic acid comprises a sequence that is at least 96% identical to SEQ ID NO: 11. According to some embodiments, the nucleic acid comprises a sequence that is at least 97% identical to SEQ ID NO:
  • the nucleic acid comprises a sequence that is at least 98% identical to SEQ ID NO: 11. According to some embodiments, the nucleic acid comprises a sequence that is at least 99% identical to SEQ ID NO: 11. According to some embodiments, the nucleic acid consists of SEQ ID NO: 11. According to some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 12. According to some embodiments, the nucleic acid comprises a sequence that is at least 90% identical to SEQ ID NO: 12. According to some embodiments, the nucleic acid comprises a sequence that is at least 95% identical to SEQ ID NO: 12. According to some embodiments, the nucleic acid comprises a sequence that is at least 96% identical to SEQ ID NO: 12. According to some embodiments, the nucleic acid comprises a sequence that is at least 97% identical to SEQ ID NO:
  • the nucleic acid comprises a sequence that is at least 98% identical to SEQ ID NO: 12. According to some embodiments, the nucleic acid comprises a sequence that is at least 99% identical to SEQ ID NO: 12. According to some embodiments, the nucleic acid consists of SEQ
  • the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 13. According to some embodiments, the nucleic acid comprises a sequence that is at least 90% identical to SEQ ID NO: 13. According to some embodiments, the nucleic acid comprises a sequence that is at least 95% identical to SEQ ID NO: 13. According to some embodiments, the nucleic acid comprises a sequence that is at least 96% identical to SEQ ID NO: 13. According to some embodiments, the nucleic acid comprises a sequence that is at least 97% identical to SEQ ID NO:
  • the nucleic acid comprises a sequence that is at least 98% identical to SEQ ID NO: 13. According to some embodiments, the nucleic acid comprises a sequence that is at least 99% identical to SEQ ID NO: 13. According to some embodiments, the nucleic acid consists of SEQ
  • the nucleic acid sequence is codon optimized for mammalian expression. According to some embodiments, the nucleic acid sequence is codon optimized for expression in human cells. According to some embodiments, the nucleic acid sequence is a cDNA sequence. According to some embodiments, the nucleic acid sequence further comprises an operably linked functionally optimized N-terminal signal sequence. According to some embodiments, the nucleic acid sequence further comprises an operably linked hemagglutinin C-terminal tag. According to some embodiments, the nucleic acid sequence further comprises an operably linked sortilin binding inhibitory
  • the nucleic acid sequence further comprises an operably linked neuron-specific human synapsin-1 promoter (hSYNl). According to some embodiments, the nucleic acid sequence further comprises an operably linked ubiquitously-active CBA promoter.
  • the nucleic acid sequence further comprises the mouse calcium/calmodulin-dependent protein kinase II (CaMKII) promoter.
  • the nucleic acid sequence further comprises the rat tubulin alpha 1 (Tal) promoter.
  • the nucleic acid sequence further comprises the Rat neuron-specific enolase (NSE) promoter.
  • the nucleic acid sequence further comprises human platelet- derived growth factor-beta chain (PDGF) promoter.
  • the nucleic acid sequence further comprises the EF1 alpha promoter.
  • any of the promoters described in the aspects and embodiments herein may further comprise an additional
  • the nucleic acid sequence further comprises an operably linked 3 ’UTR regulatory region comprising a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • the nucleic acid sequence further comprises an operably linked polyadenylation signal.
  • the polyadenylation signal is an SV40 polyadenylation signal.
  • the polyadenylation signal is a human growth hormone (hGH) polyadenylation signal.
  • the polynucleotide further comprises an operably linked N-terminal signal sequence, optionally comprising a hemagglutinin C-terminal tag or sortilin binding inhibitory (SBI) domain, operably linked to a neuron-specific human synapsin-1 promoter, mouse calcium/calmodulin-dependent protein kinase II (CaMKII) promoter, rat tubulin alpha 1 (Tal) promoter, Rat neuron-specific enolase (NSE) promoter, Human platelet-derived growth factor-beta chain (PDGF) promoter, or ubiquitously-active CBA promoter, ubiquitously-active EF1 alpha promoter, or any of the promoters set forth in any of the aspects or embodiments herein, further comprising an additional 5 ’ CAG/CMV enhancer element, operably linked to a 3 ’UTR regulatory region
  • the disclosure provides a host cell comprising the polynucleotide of any of the aspects or embodiments herein.
  • the host cell is a mammalian cell.
  • the disclosure provides a recombinant herpes simplex virus (rHSV) comprising the polynucleotide of any of the aspects or embodiments herein.
  • rHSV herpes simplex virus
  • the disclosure provides a transgene expression cassette comprising the polynucleotide of any one of the aspects or embodiments herein, and minimal regulatory elements.
  • the disclosure provides a nucleic acid vector comprising the expression cassette of claim 24.
  • the vector is an adeno-associated viral (AAV) vector.
  • the disclosure provides a host cell comprising the transgene expression cassette of any of the aspects or embodiments herein.
  • the disclosure provides an expression vector comprising the polynucleotide of any of the aspects and embodiments herein.
  • the vector is an adeno-associated vital (AAV) vector.
  • the serotype of the capsid sequence and the serotype of the ITRs of said AAV vector are independently selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.
  • the disclosure provides a recombinant adeno-associated (rAAV) expression vector comprising the polynucleotide of any of the aspects and embodiments herein, and an AAV genomic cassette.
  • rAAV adeno-associated expression vector comprising the polynucleotide of any of the aspects and embodiments herein, and an AAV genomic cassette.
  • the AAV genomic cassette is flanked by two sequence-modulated inverted terminal repeats.
  • the disclosure provides a recombinant adeno-associated (rAAV) expression vector comprising the polynucleotide of any one of the aspects and embodiments herein, operably linked to a N-terminal signal sequence, optionally comprising a hemagglutinin C- terminal tag or sortilin binding inhibitory (SBI) domain, operably linked to a neuron-specific human synapsin-1 promoter (hSYN), a mouse calcium/calmodulin-dependent protein kinase II (CaMKII) promoter, a rat tubulin alpha 1 (Tal) promoter, a rat neuron-specific enolase (NSE) promoter, a human platelet-derived growth factor-beta chain (PDGF) promoter, or ubiquitously-active CBA promoter, a ubiquitously-active EF1 alpha promoter, or any of the promoters set forth in any of the aspects or embodiments herein, further comprising an
  • the polyadenylation signal is a SV40 or human growth hormone (hGH) polyadenylation signal.
  • the promoter is optimized to drive high progranulin expression.
  • the rAAV is a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AA-9, rhAAVIO (also called AAVrhlO), AAV10, AAV11, and AAV12.
  • the rAAV is a variant or hybrid of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, rh-AAVIO, AAV10, AAV11, and AAV12.
  • the rAAV is comprised within an AAV virion.
  • the disclosure comprises an expression vector comprising AAVrhlO- hSYN-PGRNwt.
  • the disclosure provides a recombinant herpes simplex virus (rHSV) comprising the expression vector of any of the aspects or embodiments herein.
  • rHSV herpes simplex virus
  • the disclosure provides a host cell comprising the expression vector of any of the aspects and embodiments herein.
  • the host cell is a mammalian cell.
  • the disclosure provides a transgene expression cassette comprising a polynucleotide of any of the aspects and embodiments herein, and minimal regulatory elements.
  • the disclosure provides a nucleic acid vector comprising the expression cassette of any of the aspects or embodiments herein.
  • the vector is an adeno-associated viral (AAV) vector.
  • AAV adeno-associated viral
  • the disclosure provides a composition comprising a polynucleotide of any of the aspects or embodiments herein. According to some embodiments, the disclosure provides a composition comprising a host cell of any of the aspects or embodiments herein. According to some embodiments, the disclosure provides a composition comprising a recombinant herpes simplex virus (rHSV) of any of the aspects or embodiments herein. According to some embodiments, the disclosure provides a composition comprising a transgene expression cassette of any of the aspects or embodiments herein. According to some embodiments, the disclosure provides a composition comprising an expression vector of any of the aspects or embodiments herein. According to some embodiments, the composition is a pharmaceutical composition.
  • rHSV herpes simplex virus
  • the disclosure provides a method of treating a neurodegenerative disorder comprising administering the polynucleotide of any of the aspects or embodiments herein to a subject in need thereof.
  • the disclosure provides a method of treating a neurodegenerative disorder comprising administering the transgene expression cassette of any of the aspects or embodiment herein to a subject in need thereof.
  • the disclosure provides a method of treating a neurodegenerative disorder comprising administering the expression vector of any of the aspects or embodiments herein to a subject in need thereof.
  • the disclosure provides a method of treating a neurodegenerative disorder comprising administering the recombinant adeno-associated (rAAV) expression vector of any of the aspects or embodiments herein to a subject in need thereof.
  • rAAV adeno-associated
  • the disclosure provides a method of preventing a neurodegenerative disorder comprising administering the polynucleotide of any of the aspects or embodiments herein to a subject in need thereof.
  • the disclosure provides a method of preventing a neurodegenerative disorder comprising administering the transgene expression cassette of any of the aspects or embodiment herein to a subject in need thereof.
  • the disclosure provides a method of preventing a neurodegenerative disorder comprising administering the expression vector of any of the aspects or embodiments herein to a subject in need thereof.
  • the disclosure provides a method of preventing a neurodegenerative disorder comprising administering the recombinant adeno-associated (rAAV) expression vector of any of the aspects or embodiments herein to a subject in need thereof.
  • rAAV adeno-associated
  • the disclosure provides a method of treating a neurodegenerative disorder comprising administering a recombinant adeno-associated (rAAV) viral particle comprising the polynucleotide of any of the aspects or embodiments herein to a subject in need thereof.
  • the disclosure provides a method of preventing a neurodegenerative disorder comprising administering a recombinant adeno-associated (rAAV) viral particle comprising the polynucleotide of any of the aspects or embodiments herein to a subject in need thereof.
  • the neurodegenerative disorder is characterized by cognitive disruption, behavioral impairment, deficient lysosomal storage, or combination thereof.
  • the neurodegenerative disorder is a progranulin-associated neurodegenerative disorder.
  • the neurodegenerative disorder is familial frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), neuronal ceroid lipofuscinosis (NCL), or Alzheimer’s disease (AD).
  • the neuronal ceroid lipofuscinosis (NCL) is neuronal ceroid lipofuscinosis- 11 (CLN11).
  • the administration is to the central nervous system.
  • the administration is intravenous, intra cerebroventricular, intrathecal, or a combination thereof.
  • the disclosure provides a method for producing recombinant AAV viral particles comprising: co-infecting a suspension a cell with a first recombinant herpesvirus comprising a nucleic acid encoding an AAV rep and an AAV cap gene each operably linked to a promoter; and a second recombinant herpesvirus comprising a progranulin gene, and a promoter operably linked to said gene; and allowing the cell to produce the recombinant AAV viral particles, thereby producing the recombinant AAV viral particles.
  • the cap gene is selected from an AAV with a serotype selected from the group consisting of AAV-1, AAV-2, AAV -3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, rh-AAV-10, AAV11, and AAV12.
  • the first herpesvirus and the second herpesvirus are viruses selected from the group consisting of cytomegalovirus (CMV), herpes simplex (HSV) and varicella zoster (VZV) and epstein barr virus (EBV).
  • the herpesvirus is replication defective.
  • the co-infection is simultaneous.
  • FIG. 1 shows a schematic of a PGRN construct, comprising inverted terminal repeats (ITR) at each end, a human synapsin 1 (hSYNl) or chicken beta actin (CBA) promoter, PGRN optionally comprising a C-terminal HA tag or a deletion of 3-16 nucleic acids in the C-terminal, Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE), SV40 early polyadenylation signals (SV40pA) and optionally staffer DNA.
  • WPRE Woodchuck Hepatitis Virus
  • WPRE Posttranscriptional Regulatory Element
  • SV40pA SV40 early polyadenylation signals
  • optionally staffer DNA optionally staffer DNA.
  • FIG. 2 shows the nucleic acid sequence of the chicken beta actin (CBA) promoter (SEQ ID NO: 1).
  • FIG. 3 shows the nucleic acid sequence of the human synapsin 1 (hSYNl) promoter (SEQ ID NO: 2).
  • FIG. 4 shows the 5 ’-3’ inverted terminal repeat (ITR) for single stranded (ss) and self complimentary (sc) AAV genomes (SEQ ID NO: 3).
  • ITR inverted terminal repeat
  • ss single stranded
  • sc self complimentary AAV genomes
  • FIG. 5A shows the 3 ’-5’ ITR for single stranded (ss) AAV genomes only’ (SEQ ID NO: 4).
  • FIG. 5B shows the 3 ’-5’ TRS for self-complimentary (sc) AAV genomes only (SEQ ID NO: 26).
  • TRS refers to a shortened ITR.
  • FIG. 6 shows the nucleic acid sequence of wild type human progranulin (hPGRNwt) (SEQ ID NO: 5).
  • FIG. 7 shows the nucleic acid sequence of wild type mouse progranulin (mPGRNwt) (SEQ ID NO: 6). mPGRNwt is 78% similar to hPGRN.
  • FIG. 8 shows the nucleic acid sequence of macaque (Macaca mulatto) (SEQ ID NO: 7).
  • Macaque PGRN is 96% similar to hPGRN.
  • FIG. 9 shows the nucleic acid sequence of hPGRNcoA (SEQ ID NO: 8).
  • FIG. 10 shows the nucleic acid sequence of hPGRNcoB (SEQ ID NO: 9).
  • FIG. 11 shows the nucleic acid sequence of hPGRNcoC (SEQ ID NO: 10).
  • FIG. 12 shows the nucleic acid sequence of hPGRNcoD (SEQ ID NO: 11).
  • FIG. 13 shows the nucleic acid sequence of hPGRNcoE (SEQ ID NO: 12).
  • FIG. 14 shows the nucleic acid sequence of hPGRNcoF (SEQ ID NO: 13).
  • FIG. 15 shows the nucleic acid sequence of a hPGRN sortilin-binding deficient variant (SEQ ID NO: 14).
  • FIG. 16 shows the nucleic acid sequence of a hemagglutinin (HA) tag (SEQ ID NO: 15).
  • FIG. 17 shows the nucleic acid sequence of WPRE (SEQ ID NO: 16).
  • FIG. 18 shows the nucleic acid sequence of SV40 pA (SEQ ID NO: 17).
  • FIG. 19 shows the nucleic acid sequence of CaMKII promoter (364 bp) corresponding to
  • FIG. 20 shows the nucleic acid sequence of Rat tubulin alpha 1 (Tal) promoter (1034 bp) (SEQ ID NO: 19).
  • FIG. 21 shows the nucleic acid sequence of Rat neuron-specific enolase (NSE) promoter (1801 bp) (SEQ ID NO: 20).
  • FIG. 22 shows the nucleic acid sequence of a human platelet-derived growth factor-beta chain (PDGF-B) promoter (SEQ ID NO: 21 (PDGFB l), SEQ ID NO: 24 (PDG-B 2) and SEQ ID NO: 25 (PDGFB 3)).
  • PDGF-B human platelet-derived growth factor-beta chain
  • the sequence for the PDGF-B promoter includes at minimum 1 of the 3 core promoter sequences provided or any unique combination thereof. Interspersed sequences can be any non-coding, non-regulatory functional sequence, including no sequence at all.
  • FIG. 23 shows the nucleic acid sequence of EFlalpha promoter (806 bp) (SEQ ID NO: 22).
  • FIG. 24 shows the nucleic acid sequence of CAG/CMV Enhancer (SEQ ID NO: 23).
  • FIG. 25 summarizes results of rAAVrhlO, AAV9, AHSmax and AAV2tyf capsids that were tested for expression of GFP reporter in non-human primates after intrathecal injection. Percent GFP positive cells and GFP intensity in various regions of the brain are shown.
  • FIG. 26 summarizes the results of rAAVrhlO biodistribution in non-human primates by injection into the cisterna magna (ICM).
  • FIG. 27 shows graphs that depict the results of experiments testing the effects of CBA or hSYN promoters on GFP expression in HEK293 and SH-SY5Y cells.
  • FIG. 28 shows graphs that depict GFP expression after rAAVRhlO-CBA-hGFP transduction in HEK293 and SHSY-5Y cells, with and without AD5 vector.
  • FIG. 29 shows the results of an ELISA experiment to determine protein expression of the 6 PGRN codon optimized variants (designated coA-coF) compared to wild type.
  • FIG. 30 shows the results of a Western blot experiment to determine protein expression of the 6 PGRN codon optimized variants (designated coA-coF) compared to wild type.
  • FIG. 31 shows a graph that depicts the results of testing the effects of the codon optimized variants coE (PGRNcoE) and coF (PGRNcoF) under the control of hSYN promoter on progranulin expression in HEK 293 cells.
  • FIG. 32 shows the results of a Western blot experiment to confirm progranulin expression.
  • FIG. 33 shows the results of an ELISA experiment to determine protein expression of the 6 PGRN codon optimized variants (designated coA-coF) compared to wild type.
  • FIG. 34 shows progranulin expression determined after 8 and 10 weeks in an in vivo study in NHP with r AA VRh 10 -hSYN -PGRNwt.
  • administer As used herein, the terms “administer,” “administering,” “administration,” and the like, are meant to refer to methods that are used to enable delivery of therapeutics or pharmaceutical compositions to the desired site of biological action.
  • AAV virion is meant to refer broadly to a complete virus particle, such as for example a wild type AAV virion particle, which comprises single stranded genome DNA packaged into AAV capsid proteins.
  • the single stranded nucleic acid molecule is either sense strand or antisense strand, as both strands are equally infectious.
  • rAAV viral particle refers to a recombinant AAV virus particle, i.e., a particle that is infectious but replication defective.
  • a rAAV viral particle comprises single stranded genome DNA packaged into AAV capsid proteins.
  • biomass is meant to refer broadly to any apparatus that can be used for the purpose of culturing cells.
  • carrier is meant to include any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • solvents dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce a toxic, an allergic, or similar untoward reaction when administered to a host.
  • flanking refers to a relative position of one nucleic acid sequence with respect to another nucleic acid sequence.
  • B is flanked by A and C.
  • AxBxC is flanked by A and C.
  • flanking sequence precedes or follows a flanked sequence but need not be contiguous with, or immediately adjacent to the flanked sequence.
  • gene delivery means a process by which foreign DNA is transferred to host cells for applications of gene therapy.
  • the terms “gene” or “coding sequence,” is meant to refer broadly to a DNA region (the transcribed region) which encodes a protein.
  • a coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide when placed under the control of an appropriate regulatory region, such as a promoter.
  • a gene may comprise several operably linked fragments, such as a promoter, a 5’- leader sequence, a coding sequence and a 3 ’-non -translated sequence, comprising a polyadenylation site.
  • expression of a gene refers to the process wherein a gene is transcribed into an RNA and/or translated into an active protein.
  • GOI gene of interest
  • herpesvirus or “herpesviridae family, are meant to refer broadly to the general family of enveloped, double-stranded DNA viruses with relatively large genomes.
  • the family replicates in the nucleus of a wide range of vertebrate and invertebrate hosts, in preferred embodiments, mammalian hosts, for example in humans, horses, cattle, mice, and pigs.
  • Exemplary members of the herpesviridae family include cytomegalovirus (CMV), herpes simplex virus types 1 and 2 (HSV 1 and HSV2) and varicella zoster (VZV) and Epstein Barr Virus (EBV).
  • CMV cytomegalovirus
  • HSV 1 and HSV2 herpes simplex virus types 1 and 2
  • VZV varicella zoster
  • Epstein Barr Virus Epstein Barr Virus
  • heterologous means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated.
  • a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide).
  • a cellular sequence e.g., a gene or portion thereof
  • a heterologous nucleotide sequence with respect to the vector is a heterologous nucleotide sequence with respect to the vector.
  • the term “increase,” “enhance,” “raise” generally refers to the act of increasing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition.
  • the term “infection,” is meant to refer broadly to delivery of heterologous DNA into a cell by a virus.
  • co-infection means “simultaneous infection,” “double infection,” “multiple infection,” or “serial infection” with two or more viruses. Infection of a producer cell with two (or more) viruses will be referred to as “co-infection.”
  • transfection refers to a process of delivering heterologous DNA to a cell by physical or chemical methods, such as plasmid DNA, which is transferred into the cell by means of electroporation, calcium phosphate precipitation, or other methods well known in the art.
  • inverted terminal repeat or “ITR” sequence is meant to refer to relatively short sequences found at the termini of viral genomes which are in opposite orientation.
  • An “AAV inverted terminal repeat (ITR)” sequence is an approximately 145 -nucleotide sequence that is present at both termini of the native single-stranded AAV genome. The outermost nucleotides of the ITR can be present in either of two alternative orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome.
  • a “wild -type ITR” “WT-ITR” or “ITR” refers to the sequence of a naturally occurring ITR sequence in an AAV or other Dependovirus that retains, e.g., Rep binding activity and Rep nicking ability.
  • the nucleotide sequence of a WT-ITR from any AAV serotype may slightly vary from the canonical naturally occurring sequence due to degeneracy of the genetic code or drift, and therefore WT- ITR sequences encompassed for use herein include WT-ITR sequences as result of naturally occurring changes taking place during the production process (e.g., a replication error).
  • terminal repeat includes any viral terminal repeat or synthetic sequence that comprises at least one minimal required origin of replication and a region comprising a palindrome hairpin structure.
  • a Rep-binding sequence (“RBS”) also referred to as RBE (Rep-binding element)
  • RBE Rep-binding element
  • TRS terminal resolution site
  • RBS Rep-binding sequence
  • TRS terminal resolution site
  • TRs that are the inverse complement of one another within a given stretch of polynucleotide sequence are typically each referred to as an “inverted terminal repeat” or “ITR”.
  • ITRs mediate replication, virus packaging, integration and provirus rescue.
  • in vivo refers to assays or processes that occur in or within an organism, such as a multicellular animal. In some of the aspects described herein, a method or use can be said to occur “in vivo” when a unicellular organism, such as a bacterium, is used.
  • ex vivo refers to methods and uses that are performed using a living cell with an intact membrane that is outside of the body of a multicellular animal or plant, e.g., explants, cultured cells, including primary cells and cell lines, transformed cell lines, and extracted tissue or cells, including blood cells, among others.
  • in vitro refers to assays and methods that do not require the presence of a cell with an intact membrane, such as cellular extracts, and can refer to the introducing of a programmable synthetic biological circuit in a non-cellular system, such as a medium not comprising cells or cellular systems, such as cellular extracts.
  • the term “isolated” molecule e.g., an isolated nucleic acid or protein or cell
  • the term “minimal regulatory elements” is meant to refer to regulatory elements that are necessary for effective expression of a gene in a target cell and thus should be included in a transgene expression cassette.
  • Such sequences could include, for example, promoter or enhancer sequences, a polylinker sequence facilitating the insertion of a DNA fragment within a plasmid vector, and sequences responsible for intron splicing and polyadenlyation of mRNA transcripts.
  • the term “minimize”, “reduce”, “decrease,” and/or “inhibit” generally refers to the act of reducing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition.
  • 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 tissue of the brain and spinal cord of a vertebrate.
  • the term “peripheral nervous system” refers to all cells and tissue of the portion of the nervous system outside the brain and spinal cord.
  • the term "nervous system” includes, but is not limited to, neuronal cells, glial cells, astrocytes, cells in the cerebrospinal fluid (CSF), cells in the interstitial spaces, cells in the protective coverings of the spinal cord, epidural cells (i.e., cells outside of the dura mater), cells in non-neural tissues adjacent to or in contact with or innervated by neural tissue, cells in the epineurium, perineurium, endoneurium, funiculi, fasciculi, and the like.
  • CSF cerebrospinal fluid
  • epidural cells i.e., cells outside of the dura mater
  • neural tissue i.e., cells outside of the dura mater
  • non-naturally occurring is meant to refer broadly to a protein, nucleic acid, ribonucleic acid, or virus that does not occur in nature. For example, it may be a genetically modified variant, e.g., cDNA or codon-optimized nucleic acid.
  • a “nucleic acid” or a “nucleic acid molecule” is meant to refer to a molecule composed of chains of monomeric nucleotides, such as, for example, DNA molecules (e.g., cDNA or genomic DNA).
  • a nucleic acid may encode, for example, a promoter, the PGRN gene or portion thereof, or regulatory elements.
  • a nucleic acid molecule can be single-stranded or double-stranded.
  • a “PGRN nucleic acid” refers to a nucleic acid that comprises the PGRN gene or a portion thereof, or a functional variant of the PGRN gene or a portion thereof.
  • a functional variant of a gene includes a variant of the gene with minor variations such as, for example, silent mutations, single nucleotide polymorphisms, missense mutations, and other mutations or deletions that do not significantly alter gene function.
  • the asymmetric ends of DNA and RNA strands are called the 5' (five prime) and 3' (three prime) ends, with the 5' end having a terminal phosphate group and the 3' end a terminal hydroxyl group.
  • the five prime (5’) end has the fifth carbon in the sugar-ring of the deoxyribose or ribose at its terminus.
  • Nucleic acids are synthesized in vivo in the 5'- to 3'-direction, because the polymerase used to assemble new strands attaches each new nucleotide to the 3 '-hydroxyl (-OH) group via a phosphodiester bond.
  • nucleic acid construct refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic.
  • nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present disclosure.
  • a DNA sequence that “encodes” a particular PGRN protein is a nucleic acid sequence that is transcribed into the particular RNA and/or protein.
  • a DNA polynucleotide may encode an RNA (mRNA) that is translated into protein, or a DNA polynucleotide may encode an RNA that is not translated into protein (e.g., tRNA, rRNA, or a DNA-targeting RNA; also called “non-coding" RNA or "ncRNA”).
  • operatively linked or “operably linked” or “coupled” can refer to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in an expected manner.
  • a promoter can be operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.
  • a “percent (%) sequence identity” with respect 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 with the amino acid residues or nucleotides in the reference polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Alignment for purposes of determining percent amino acid or nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Supp. 30, section 7.7.18, Table 7.7.1, and including BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • An example of an alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
  • the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows: 100 times the fraction W/Z, where W is the number of nucleotides scored as identical matches by the sequence alignment program in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.
  • composition is meant to refer to a composition or agent described herein (e.g . a recombinant adeno-associated (rAAV) expression vector), optionally mixed with at least one pharmaceutically acceptable chemical component, such as, though not limited to carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, excipients and the like.
  • pharmaceutically acceptable chemical component such as, though not limited to carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, excipients and the like.
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length.
  • polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • a "polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • progranulin As used herein, the terms “progranulin”, “PGRN”, “granulin-epithelin precursor”, “GEP”, “PC- cell-derived growth factor”, “PCDGF”, “proepithelin”, “acrogranin”, and “GP80” may be used herein interchangeably.
  • a progranulin (PGRN) or “a progranulin (PGRN) nucleic acid” refers to a nucleic acid selected from a nucleic acid comprising SEQ ID NO: 5, a nucleic acid with about 95% homology, about 96%, about 97%, about 98%, about 99% homology with SEQ ID NO: 5, a nucleic acid consisting of SEQ ID NO: 5; a nucleic acid comprising SEQ ID NO: 6, a nucleic acid with about 95% homology, about 96%, about 97%, about 98%, about 99% homology with SEQ ID NO: 6, a nucleic acid consisting of SEQ ID NO: 6; a nucleic acid comprising SEQ ID NO: 7, a nucleic acid with about 95% homology, about 96%, about 97%, about 98%, about 99% homology with SEQ ID NO: 7, a nucleic acid consisting of SEQ ID NO: 7; a nucleic acid comprising SEQ ID NO NO: 7;
  • a “promoter” is meant to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence.
  • the enzyme that synthesizes RNA known as RNA polymerase, attaches to the DNA near a gene.
  • Promoters contain specific DNA sequences and response elements that provide an initial binding site for RNA polymerase and for transcription factors that recruit RNA polymerase. According to some embodiments, the promoter is highly specific for transgene expression in cells of the CNS.
  • the promoter is highly specific for neuron-specific transgene expression.
  • the promoter is an endogenous PGRN promoter.
  • the promoter is a chicken beta-actin (CBA) promoter.
  • the promoter is a human synapsin-1 gene promoter (hSynl) promoter.
  • FIG. 3 shows the nucleic acid sequence of the human synapsin 1 (hSYNl) promoter (SEQ ID NO: 2).
  • the hSYNl promoter comprises SEQ ID NO: 2.
  • the hSYNl promoter consists of SEQ ID NO: 2.
  • a CBA promoter refers to a polynucleotide sequence derived from a chicken beta-actin gene (e.g., Gallus gallus beta actin, represented by GenBank Entrez Gene ID 396526).
  • FIG. 2 shows the nucleic acid sequence of the chicken beta actin (CBA) promoter (SEQ ID NO: 1).
  • the CBA promoter comprises SEQ ID NO: 1.
  • the CBA promoter consists of SEQ ID NO: 1.
  • the promoter is a mouse calcium/calmodulin-dependent protein kinase II (CaMKII) promoter.
  • the mouse calcium/calmodulin-dependent protein kinase II promoter is a moderate strength neuron-specific promoter.
  • the CaMKII promoter comprises SEQ ID NO: 18.
  • the CaMKII promoter consists of SEQ ID NO: 18.
  • the promoter is a rat tubulin alpha 1 (Tal) promoter.
  • the rat tubulin alpha 1 promoter is a moderate strength promoter responsible for regulating neuron gene expression as a function of morphological growth.
  • the rat Tal promoter is described in Gloster et al. 1994 J of Neuroscience, incorporated by reference in its entirety herein.
  • FIG. 20 shows the nucleic acid sequence of Rat tubulin alpha 1 (Tal) promoter (SEQ ID NO: 19).
  • the Tal promoter comprises SEQ ID NO: 19. According to some embodiments, the Tal promoter consists of SEQ ID NO: 19. According to some embodiments, the promoter is a rat neuron-specific enolase (NSE) promoter. The rat neuron-specific enolase promoter is a moderate strength, developmentally regulated promoter.
  • FIG. 21 shows the nucleic acid sequence of Rat neuron-specific enolase (NSE) promoter (SEQ ID NO: 20). According to some embodiments, the NSE promoter comprises SEQ ID NO: 20. According to some embodiments, the NSE promoter consists of SEQ ID NO: 20.
  • the promoter is a human platelet-derived growth factor-beta chain (PDGF) promoter.
  • the human platelet-derived growth factor- beta chain promoter is a moderate strength promoter specific to neurons and (neuron-associated) glial cells.
  • FIG. 22 shows the nucleic acid sequence of a human platelet-derived growth factor-beta chain (PDGF) promoter (SEQ ID NO: 21, SEQ ID NO: 24 and SEQ ID NO: 25).
  • the PDGF-beta chain promoter comprises SEQ ID NO: 21. According to some embodiments, the PDGF-beta chain promoter consists of SEQ ID NO: 21. According to some embodiments, the promoter is an ubiquitously-active EFlalpha promoter.
  • the EFla promoter is a strong ubiquitous promoter of mammalian origin that expresses in most cell types, including cells of the CNS.
  • FIG. 23 shows the nucleic acid sequence of EFlalpha promoter (SEQ ID NO: 22). According to some embodiments, the EFlalpha promoter comprises SEQ ID NO: 22. According to some embodiments, the EFlalpha promoter consists of SEQ ID NO: 22.
  • any of the promoters set forth in the aspects and embodiments herein further comprise an additional 5’ CAG/CMV enhancer element.
  • the CAG/CMV enhancer is a strong enhancer element derived from the enhancer that affects the strong ubiquitous CAG promoter and it’s CMV parent promoter; both are commonly employed to enhance transcriptional activity of core promoters.
  • FIG. 24 shows the nucleic acid sequence of a CAG/CMV enhancer (SEQ ID NO: 23).
  • a promoter can be said to drive expression or drive transcription of the nucleic acid sequence that it regulates.
  • the phrases “operably linked,” “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” indicate that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence it regulates to control transcriptional initiation and/or expression of that sequence.
  • An “inverted promoter,” as used herein, refers to a promoter in which the nucleic acid sequence is in the reverse orientation, such that what was the coding strand is now the non-coding strand, and vice versa. Inverted promoter sequences can be used in various embodiments to regulate the state of a switch.
  • a promoter can be used in conjunction with an enhancer.
  • a promoter can be one naturally associated with a gene or sequence, as can be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon of a given gene or sequence. Such a promoter can be referred to as “endogenous.”
  • an enhancer can be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • a coding nucleic acid segment is positioned under the control of a “recombinant promoter” or “heterologous promoter,” both of which refer to a promoter that is not normally associated with the encoded nucleic acid sequence it is operably linked to in its natural environment.
  • a recombinant or heterologous enhancer refers to an enhancer not normally associated with a given nucleic acid sequence in its natural environment.
  • Such promoters or enhancers can include promoters or enhancers of other genes; promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell; and synthetic promoters or enhancers that are not “naturally occurring,” i.e., comprise different elements of different transcriptional regulatory regions, and/or mutations that alter expression through methods of genetic engineering that are known in the art.
  • Enhancer refers to a cis-acting regulatory sequence (e.g., 50-1,500 base pairs) that binds one or more proteins (e.g., activator proteins, or transcription factor) to increase transcriptional activation of a nucleic acid sequence. Enhancers can be positioned up to 1,000,000 base pars upstream of the gene start site or downstream of the gene start site that they regulate.
  • the term “recombinant” can refer to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature.
  • the term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and/or mRNAs encoded by such nucleic acids.
  • rHSV herpes simplex virus type 1
  • rHSV-rep2cap2 or “rHSV- rep2capl” is meant an rHSV in which the AAV rep and cap genes from either AAV serotype 1 or 2 have been incorporated into the rHSV genome, in certain embodiments, a DNA sequence encoding a therapeutic gene of interest has been incorporated into the viral genome.
  • a “subject” or “patient” or “individual” to be treated by the method of the invention is meant to refer to either a human or non-human animal.
  • a “nonhuman animal” includes any vertebrate or invertebrate organism.
  • a human subject can be of any age, gender, race or ethnic group, e.g., Caucasian (white), Asian, African, black, African American, African European, Hispanic, Middle eastern, etc.
  • the subject can be a patient or other subject in a clinical setting.
  • the subject is already undergoing treatment.
  • the subject is a neonate, infant, child, adolescent, or adult.
  • therapeutic effect refers to a consequence of treatment, the results of which are judged to be desirable and beneficial.
  • a therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation.
  • a therapeutic effect can also include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation.
  • therapeutically effective amount may be initially determined from preliminary in vitro studies and/or animal models.
  • a therapeutically effective dose may also be determined from human data.
  • the applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan.
  • substitution mutation profile is meant to refer to a panel of conservative amino acid substitutions in the inter-domain regions of progranulin that eliminate one or more of its 5 elastase cleavage sites and/or 2 other proteolytic cleavage sites (see e.g., Cenik et al, JBC 2012; Zhu et al. , Cell 2002, both of which are incorporated by reference in their entireties herein).
  • the substitution mutations will reduce or prevent PGRN from being processed into granulins.
  • transgene is meant to refer to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and optionally, translated and/or expressed under appropriate conditions. In aspects, it confers a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic or diagnostic outcome.
  • transgene expression cassette or “expression cassette” are used interchangeably and refer to a linear stretch of nucleic acids that includes a transgene that is operably linked to one or more promoters or other regulatory sequences sufficient to direct transcription of the transgene, but which does not comprise capsid-encoding sequences, other vector sequences or inverted terminal repeat regions.
  • An expression cassette may additionally comprise one or more cis-acting sequences (e.g., promoters, enhancers, or repressors), one or more introns, and one or more post- transcriptional regulatory elements.
  • a transgene expression cassette comprises the gene sequences that a nucleic acid vector is to deliver to target cells. These sequences include the gene of interest (e.g., PGRN nucleic acids or variants thereof), one or more promoters, and minimal regulatory elements.
  • treatment or “treating” a disease or disorder are meant to refer to alleviation of one or more signs or symptoms of the disease or disorder, diminishment of extent of disease or disorder, stabilized (e.g., not worsening) state of disease or disorder, preventing spread of disease or disorder, delay or slowing of disease or disorder progression, amelioration or palliation of the disease or disorder state, and remission (whether partial or total), whether detectable or undetectable.
  • PGRN when expressed in an effective amount (or dosage) is sufficient to prevent, correct, and/or normalize an abnormal physiological response, e.g., a therapeutic effect that is sufficient to reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant feature of disease or disorder.
  • Treatment can also refer to prolonging survival as compared to expected survival if not receiving treatment.
  • vector is meant to refer to a recombinant plasmid or virus that comprises a nucleic acid to be delivered into a host cell, either In vitro or in vivo.
  • expression vector refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector.
  • the sequences expressed will often, but not necessarily, be heterologous to the cell.
  • An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.
  • expression refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing.
  • “Expression products” include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene.
  • the term “gene” means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences.
  • the gene may or may not include regions preceding and following the coding region, e.g., 5’ untranslated (5’UTR) or “leader” sequences and 3’ UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons).
  • the term “recombinant viral vector” is meant to refer to a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of viral origin).
  • the recombinant nucleic acid is flanked by at least one inverted terminal repeat sequence (ITR).
  • ITR inverted terminal repeat sequence
  • the recombinant nucleic acid is flanked by two ITRs.
  • rAAV vector recombinant AAV vector
  • rAAV vector a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of AAV origin) that are flanked by at least one AAV inverted terminal repeat sequence (ITR).
  • ITR AAV inverted terminal repeat sequence
  • rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e. AAV Rep and Cap proteins).
  • a rAAV vector When a rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), then the rAAV vector may be referred to as a "pro-vector" which can be "rescued” by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions.
  • a rAAV vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral particle, e.g., an AAV particle.
  • a rAAV vector can be packaged into an AAV virus capsid to generate a "recombinant adeno-associated viral particle (rAAV particle)".
  • rAAV virus or “rAAV viral particle” is meant to refer to a viral particle composed of at least one AAV capsid protein and an encapsidated rAAV vector genome.
  • reporter refer to proteins that can be used to provide detectable read-outs. Reporters generally produce a measurable signal such as fluorescence, color, or luminescence. Reporter protein coding sequences encode proteins whose presence in the cell or organism is readily observed. For example, fluorescent proteins cause a cell to fluoresce when excited with light of a particular wavelength, luciferases cause a cell to catalyze a reaction that produces light, and enzymes such as b-galactosidase convert a substrate to a colored product.
  • reporter polypeptides useful for experimental or diagnostic purposes include, but are not limited to b-lactamase, b -galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP) and other fluorescent proteins, chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.
  • Transcriptional regulators refer to transcriptional activators and repressors that either activate or repress transcription of a gene of interest, such as PGRN. Promoters are regions of nucleic acid that initiate transcription of a particular gene Transcriptional activators typically bind nearby to transcriptional promoters and recruit RNA polymerase to directly initiate transcription. Repressors bind to transcriptional promoters and sterically hinder transcriptional initiation by RNA polymerase. Other transcriptional regulators may serve as either an activator or a repressor depending on where they bind and cellular and environmental conditions. Non-limiting examples of transcriptional regulator classes include, but are not limited to homeodomain proteins, zinc-finger proteins, winged-helix (forkhead) proteins, and leucine- zipper proteins.
  • a “repressor protein” or “inducer protein” is a protein that binds to a regulatory sequence element and represses or activates, respectively, the transcription of sequences operatively linked to the regulatory sequence element.
  • Preferred repressor and inducer proteins as described herein are sensitive to the presence or absence of at least one input agent or environmental input.
  • Preferred proteins as described herein are modular in form, comprising, for example, separable DNA-binding and input agent-binding or responsive elements or domains.
  • the term “consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.
  • the use of “comprising” indicates inclusion rather than limitation.
  • the term “consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.
  • nucleic acid molecules for potential therapeutic use are provided herein.
  • the present disclosure provides promoters, expression cassettes, vectors, kits, and methods that can be used in the treatment of neurodegenerative diseases or disorders.
  • Certain aspects of the disclosure relate to delivering a heterologous nucleic acid to a subject comprising administering a recombinant adeno-associated virus (rAAV) vector.
  • rAAV recombinant adeno-associated virus
  • the disclosure provides methods of treating or preventing neurodegenerative diseases or disorders comprising delivery of a composition comprising rAAV vectors described herein to the subject, wherein the rAAV vector comprises a heterologous nucleic acid (e.g . a nucleic acid encoding PGRN).
  • An object of the present disclosure is to deliver nucleic acids encoding PGRN to the central nervous system (CNS) with successful expression in the CNS, and the treatment of neurodegenerative disease.
  • the expressed PGRN protein is functional for the treatment of treatment of a neurodegenerative disease or disorders.
  • expressed PGRN protein does not cause an immune system reaction.
  • PGRN is a glycoprotein encoded by the GRN/Gm gene with multiple cellular functions, including neurotrophic, anti-inflammatory and lysosome regulatory properties. Mutations in the GRN gene can lead to frontotemporal lobar degeneration (FTD), a cause of dementia, and neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Both diseases are associated with loss of PGRN function resulting, amongst other features, in enhanced microglial neuroinflammation and lysosomal dysfunction. PGRN has also been implicated in Alzheimer’s disease (AD). Mendsaikhan el al. Cells.
  • AD Alzheimer’s disease
  • the gene of interest e.g ., PGRN
  • PGRN the gene of interest
  • the gene of interest is optimized to be superior in expression (and/or function) to wildtype PGRN, and further has the ability to discriminate (at the DNA RNA level) from wildtype PGRN.
  • the gene of interest e.g., PGRN
  • PGRN the gene of interest
  • PGRN the gene of interest
  • PGRN C-terminal motif PGRN (589-593) LRQLL, is essential for SORT 1 -mediated endocytosis (Zheng et al., PLoS One. 2011;6(6):e21023).
  • the gene of interest (e.g., PGRN) is optimized to generate less granulin products.
  • a “PGRN nucleic acid” refers to a nucleic acid that comprises the PGRN gene or a portion thereof, or a functional variant of the PGRN gene or a portion thereof.
  • a functional variant of a gene includes a variant of the gene with minor variations such as, for example, silent mutations, single nucleotide polymorphisms, missense mutations, and other mutations or deletions that do not significantly alter gene function.
  • the nucleic acid encoding the PGRN protein is 1779 bp in length.
  • the nucleic acid comprises SEQ ID NO: 5.
  • the nucleic acid is at least 85% identical to SEQ ID NO: 5.
  • the nucleic acid is at least 90% identical to SEQ ID NO: 5.
  • the nucleic acid is at least 95% identical to SEQ ID NO: 5.
  • the nucleic acid is at least 96% identical to SEQ ID NO: 5.
  • the nucleic acid is at least 96% identical to SEQ ID NO: 5.
  • the nucleic acid is at least 97% identical to SEQ ID NO: 5.
  • the nucleic acid is at least 98% identical to SEQ ID NO: 5. According to one embodiment, the nucleic acid is at least 99% identical to SEQ ID NO: 5. According to one embodiment, the nucleic acid consists of SEQ ID NO: 5.
  • the nucleic acid encoding the PGRN protein is 1767 bp in length.
  • the nucleic acid comprises SEQ ID NO: 6.
  • the nucleic acid is at least 85% identical to SEQ ID NO: 6.
  • the nucleic acid is at least 90% identical to SEQ ID NO: 6.
  • the nucleic acid is at least 95% identical to SEQ ID NO: 6.
  • the nucleic acid is at least 96% identical to SEQ ID NO: 6.
  • the nucleic acid is at least 97% identical to SEQ ID NO: 6.
  • the nucleic acid is at least 98% identical to SEQ ID NO: 6.
  • the nucleic acid is at least 99% identical to SEQ ID NO: 6.
  • the nucleic acid consists of SEQ ID NO: 6.
  • the nucleic acid encoding the PGRN protein is 1779 bp in length.
  • the nucleic acid comprises SEQ ID NO: 7.
  • the nucleic acid is at least 85% identical to SEQ ID NO: 7.
  • the nucleic acid is at least 90% identical to SEQ ID NO: 7.
  • the nucleic acid is at least 95% identical to SEQ ID NO: 7.
  • the nucleic acid is at least 96% identical to SEQ ID NO: 7.
  • the nucleic acid is at least 97% identical to SEQ ID NO: 7.
  • the nucleic acid is at least 98% identical to SEQ ID NO: 7.
  • the nucleic acid is at least 99% identical to SEQ ID NO: 7.
  • the nucleic acid consists of SEQ ID NO: 7.
  • the nucleic acid encoding the PGRN protein is 1779 bp in length.
  • the nucleic acid comprises SEQ ID NO: 8.
  • the nucleic acid is at least 85% identical to SEQ ID NO: 8.
  • the nucleic acid is at least 90% identical to SEQ ID NO: 8.
  • the nucleic acid is at least 95% identical to SEQ ID NO: 8.
  • the nucleic acid is at least 96% identical to SEQ ID NO: 8.
  • the nucleic acid is at least 97% identical to SEQ ID NO: 8.
  • the nucleic acid is at least 98% identical to SEQ ID NO: 8.
  • the nucleic acid is at least 99% identical to SEQ ID NO: 8.
  • the nucleic acid consists of SEQ ID NO: 8.
  • the nucleic acid encoding the PGRN protein is 1779 bp in length.
  • the nucleic acid comprises SEQ ID NO: 9.
  • the nucleic acid is at least 85% identical to SEQ ID NO: 9.
  • the nucleic acid is at least 90% identical to SEQ ID NO: 9.
  • the nucleic acid is at least 95% identical to SEQ ID NO: 9.
  • the nucleic acid is at least 96% identical to SEQ ID NO: 9.
  • the nucleic acid is at least 97% identical to SEQ ID NO: 9.
  • the nucleic acid is at least 98% identical to SEQ ID NO: 9.
  • the nucleic acid is at least 99% identical to SEQ ID NO: 9.
  • the nucleic acid consists of SEQ ID NO: 9.
  • the nucleic acid encoding the PGRN protein is 1779 bp in length.
  • the nucleic acid comprises SEQ ID NO: 10.
  • the nucleic acid is at least 85% identical to SEQ ID NO: 10.
  • the nucleic acid is at least 90% identical to SEQ ID NO: 10.
  • the nucleic acid is at least 95% identical to SEQ ID NO: 10.
  • the nucleic acid is at least 96% identical to SEQ ID NO: 10.
  • the nucleic acid is at least 97% identical to SEQ ID NO: 10.
  • the nucleic acid is at least 98% identical to SEQ ID NO: 10.
  • the nucleic acid is at least 99% identical to SEQ ID NO: 10.
  • the nucleic acid consists of SEQ ID NO: 10.
  • the nucleic acid encoding the PGRN protein is 1779 bp in length.
  • the nucleic acid comprises SEQ ID NO: 11.
  • the nucleic acid is at least 85% identical to SEQ ID NO: 11.
  • the nucleic acid is at least 90% identical to SEQ ID NO: 11.
  • the nucleic acid is at least 95% identical to SEQ ID NO: 11.
  • the nucleic acid is at least 96% identical to SEQ ID NO: 11.
  • the nucleic acid is at least 97% identical to SEQ ID NO: 11.
  • the nucleic acid is at least 98% identical to SEQ ID NO: 11.
  • the nucleic acid is at least 99% identical to SEQ ID NO: 8.
  • the nucleic acid consists of SEQ ID NO: 11.
  • the nucleic acid encoding the PGRN protein is 1779 bp in length.
  • the nucleic acid comprises SEQ ID NO: 12.
  • the nucleic acid is at least 85% identical to SEQ ID NO: 12.
  • the nucleic acid is at least 90% identical to SEQ ID NO: 12.
  • the nucleic acid is at least 95% identical to SEQ ID NO: 12.
  • the nucleic acid is at least 96% identical to SEQ ID NO: 12.
  • the nucleic acid is at least 97% identical to SEQ ID NO: 12.
  • the nucleic acid is at least 98% identical to SEQ ID NO: 12.
  • the nucleic acid is at least 99% identical to SEQ ID NO: 12.
  • the nucleic acid consists of SEQ ID NO: 12.
  • the nucleic acid encoding the PGRN protein is 1779 bp in length.
  • the nucleic acid comprises SEQ ID NO: 13.
  • the nucleic acid is at least 85% identical to SEQ ID NO: 13.
  • the nucleic acid is at least 90% identical to SEQ ID NO: 13.
  • the nucleic acid is at least 95% identical to SEQ ID NO: 13.
  • the nucleic acid is at least 96% identical to SEQ ID NO: 13.
  • the nucleic acid is at least 97% identical to SEQ ID NO: 13.
  • the nucleic acid is at least 98% identical to SEQ ID NO: 13.
  • the nucleic acid is at least 99% identical to SEQ ID NO: 13.
  • the nucleic acid consists of SEQ ID NO: 13.
  • the nucleic acid encoding the PGRN protein has a deletion of 3-16 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) amino acids from the C-terminus.
  • the deletion in the C-terminus of PGRN results in inhibition of PGRN sortilin binding and subsequent processing to individual granulins.
  • the nucleic acid encoding the PGRN protein consists of SEQ ID NO: 1 with a deletion of 3-16 (e.g., 3, 4, 5, 6, 7, 8, 9, 10,
  • a polynucleotide encoding a site-directed polypeptide can be codon-optimized according to methods standard in the art for expression in the cell containing the target DNA of interest. For example, if the intended target nucleic acid is in a human cell, a human codon-optimized polynucleotide encoding PGRN is contemplated for use in the constructs described herein.
  • the nucleic acid sequence is codon optimized for mammalian expression.
  • the disclosure generally provides methods for producing recombinant adeno-associated virus (AAV) viral particles comprising a PGRN gene construct and their use in methods of gene therapy for neurodegenerative diseases , and in particular neurodegenerative diseases characterized by partial or complete PGRN deficiency, e.g., frontotemporal dementia (FTD).
  • AAV vectors as described herein are particularly efficient at delivering nucleic acids (e.g., PGRN gene construct) to cells of the CNS, and in particular to neuronal cells.
  • rAAV recombinant adeno-associated virus
  • PGRN cDNA and associated genetic elements for use in recombinant adeno-associated virus (rAAV)-based gene therapy for neurodegenerative diseases, including the treatment and/or prevention of FTD, are described herein.
  • Recombinant adeno-associated virus (rAAV) vector can efficiently accommodate both PGRN target gene and associated genetic elements.
  • such vectors can be designed to specifically express PGRN in therapeutically relevant cells of the CNS.
  • the disclosure describes a method to create, evaluate, and utilize rAAV therapeutic vectors able to efficiently deliver the functional PGRN gene to patients.
  • the PGRN gene construct may comprise: (1) a 1.8 kilobase (kb) non-naturally occurring codon- optimized PGRN cDNA sequence, derived from either human, mouse, or macaque, which may possess resistance to proteolytic cleavage to granulins by means of either (a) a substitution mutation profile or by a 3-16 C-terminal amino acid deletion (to inhibit sortilin binding and subsequent processing to individual granulins), with or without a 27-nucleotide hemagglutinin C-terminal tag, (2) a 0.5 kb non-naturally occurring neuron-specific human synapsin-1 promoter (hSYNl) or 0.364 kb mouse calcium/calmodulin- dependent protein kinase II (CaMKII) promoter, or 1.034 kb rat tubulin alpha 1 (Tal) promoter, or 1.81 kb rat neuron-specific enolase (NSE) promoter, or
  • AAV Adeno-Associated Virus
  • Adeno-Associated Virus is a non-pathogenic single-stranded DNA parvovirus.
  • AAV has a capsid diameter of about 20 nm.
  • Each end of the single-stranded DNA genome contains an inverted terminal repeat (ITR), which is the only cis-acting element required for genome replication and packaging.
  • the AAV genome carries two viral genes: rep and cap.
  • the virus utilizes two promoters and alternative splicing to generate four proteins necessary for replication (Rep 78, Rep 68, Rep 52 and Rep 40).
  • a third promoter generates the transcript for three structural viral capsid proteins, 1, 2 and 3 (VP1, VP2 and VP3), through a combination of alternate splicing and alternate translation start codons. Bems & Linden Bioessays 1995; 17:237-45.
  • the three capsid proteins share the same C-terminal 533 amino acids, while VP2 and VP1 contain additional N-terminal sequences of 65 and 202 amino acids, respectively
  • the AAV virion contains a total of 60 copies of VP1 , VP2, and VP3 at a 1 : 1 :20 ratio, arranged in a T-l icosahedral symmetry. Rose el al. J Virol. 1971; 8:766-70.
  • AAV requires Adenovirus (Ad), Herpes Simplex Virus (HSV) or other viruses as a helper virus to complete its lytic life-cycle. Atchison et al. Science, 1965; 149:754-6; Hoggan et al. Proc Natl Acad Sci USA, 1966; 55:1467-74.
  • HSV Herpes Simplex Virus
  • wild-type AAV establishes latency by integration with the assistance of Rep proteins through the interaction of the ITR with the chromosome. Bems & Linden (1995).
  • AAV serotypes There are a number of different AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrhlO, AAV2.retro, and variants or hybrids thereof (e.g. AAV2 variants with HSPG mutation, AAV 1+9 hybrids).
  • AAV1 and AAV6 are two serotypes that, are efficient for the transduction of skeletal muscle. Gao, et al.
  • AAV2, AAV4, and AAV5 transduce different types of cells in the central nervous system. Davidson, et al. Proc Natl Acad Sci USA. 2000; 97:3428-3432. AAV8 and AAV5 can transduce liver cells better than AAV -2. AAV-5 based vectors transduced certain cell types (cultured airway epithelial cells, cultured striated muscle cells and cultured human umbilical vein endothelial cells) at a higher efficiency than AAV2, while both AAV2 and AAV5 showed poor transduction efficiencies for NIH 3T3, skbr3 and t-47D cell lines. Gao, et al. Proc Natl Acad Sci USA.
  • AAV1 AAV2, AAV4, AAV5, AAV8, and AAV9 show tropism for CNS tissues.
  • AAV1, AAV8, and AAV9 show tropism for heart tissues.
  • AAV2 exhibits tropism for kidney tissue.
  • AAV7, AAV8, and AAV9 exhibit tropism for liver tissue.
  • AAV4, AAV5, AAV6, and AAV9 exhibits tropism for lung tissue.
  • AAV8 exhibits tropism for pancreas cells.
  • AAV3, AAV5, and AAV8 show tropism for photoreceptor cells.
  • AAV1, AAV2, AAV4, AAV5, and AAV8 exhibit tropism for retinal pigment epithelium (RPE) cells.
  • RPE retinal pigment epithelium
  • AAV1, AAV6, AAV7, AAV8, and AAV9 show tropism for skeletal muscle.
  • AAV2 variants with heparin sulfate proteoglycan (HSPG) mutation have been shown to have enhanced neural and brain transduction.
  • Further modification to the virus can be performed to enhance the efficiency of gene transfer, for example, by improving the tropism of each serotype.
  • One approach is to swap domains from one serotype capsid to another, and thus create hybrid vectors with desirable qualities from each parent.
  • the viral capsid is responsible for cellular receptor binding, the understanding of viral capsid domain(s) critical for binding is important. Mutation studies on the viral capsid (mainly on AAV2) performed before the availability of the crystal structure were mostly based on capsid surface functionalization by adsorption of exogenous moieties, insertion of peptide at a random position, or comprehensive mutagenesis at the amino acid level. Choi, et al. Curr Gene Ther. 2005 June; 5(3): 299-310, describe different approaches and considerations for hybrid serotypes.
  • Capsids from other AAV serotypes offer advantages in certain in vivo applications over rAAV vectors based on the AAV2 capsid.
  • the appropriate use of rAAV vectors with particular serotypes may increase the efficiency of gene delivery in vivo to certain target cells that are poorly infected, or not infected at all, by AAV2 based vectors.
  • recombinant HSV vectors similar to rHSV but encoding the cap genes from other AAV serotypes, e.g., AAV1, AAV2, AAV3, AAV5 to AAV9 is achievable using the methods described herein to produce rHSV
  • recombinant AAV vectors constructed using cap genes from different AAV are preferred.
  • the significant advantages of construction of these additional rHSV vectors are ease and savings of time, compared with alternative methods used for the large-scale production of rAAV. In particular, the difficult process of constructing new rep and cap inducible cell lines for each different capsid serotypes is avoided.
  • the production, purification, and characterization of the rAAV vectors of the present invention may be carried out using any of the many methods known in the art.
  • Clark RK Recent advances in recombinant adeno-associated virus vector production. Kidney Int. 61s:9-15 (2002); Choi VW et al, Production of recombinant adeno-associated viral vectors for In vitro and in vivo use. Current Protocols in Molecular Biology 16.25.1-16.25.24 (2007) (hereinafter Choi et al.), ⁇ Grieger JC & Samulski RJ, Adeno-associated virus as a gene therapy vector: Vector development, production, and clinical applications.
  • AAV vector production may be accomplished by cotransfection of packaging plasmids. Heilbronn.
  • the cell line supplies the deleted AAV genes rep and cap and the required helper virus functions.
  • the adenovirus helper genes, VA-RNA, E2A and E4 are transfected together with the AAV rep and cap genes, either on two separate plasmids or on a single helper construct.
  • These packaging plasmids are typically transfected into 293 cells, a human cell line that constitutively expresses the remaining required Ad helper genes, E1A and E1B.
  • AAV vectors of the present invention may comprise capsid sequences derived from AAVs of any known serotype.
  • a “known serotype” encompasses capsid mutants that can be produced using methods known in the art. Such methods, include, for example, genetic manipulation of the viral capsid sequence, domain swapping of exposed surfaces of the capsid regions of different serotypes, and generation of AAV chimeras using techniques such as marker rescue. See Bowles et al.
  • the AAV vectors of the present invention may comprise ITRs derived from AAVs of any known serotype.
  • the ITRs are derived from one of the human serotypes AAV1-AAV12.
  • a pseudotyping approach is employed, wherein the genome of one ITR serotype is packaged into a different serotype capsid.
  • the capsid sequences are derived from one of the human serotypes AAV1-AAV12. According to some embodiments, the capsid sequences are derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVrh8, AAVrhlO, AAV2.retro, and variants or hybrids thereof (e.g. AAV2 variants with HSPG mutation, (AAV2-HBKO, AAVT-TT, AAV44.9), AAV 1+9 hybrids). According to some embodiments, the particular capsid sequences confer enhanced neural and brain transduction.
  • the capsid sequences are derived from an AAV2 variant with high tropism for targeting cells of the CNS (e.g., neuronal cells, astrocytes).
  • the AAV is AAV9.
  • the AAV is AAVrhlO.
  • AAV tropism is determined by the specific interaction between distinct viral capsid proteins and their cognate cellular receptors.
  • a rAAV having a capsid appropriate for the tissue being targeted can be selected.
  • recombinant AAV vectors can be directly targeted by genetic manipulation of the viral capsid sequence, particularly in the looped out region of the AAV three- dimensional structure, or by domain swapping of exposed surfaces of the capsid regions of different serotypes, or by generation of AAV chimeras using techniques such as marker rescue. See Bowles et al. Marker rescue of adeno-associated virus (AAV) capsid mutants: A novel approach for chimeric AAV production. Journal of Virology, 77(1): 423-432 (2003), as well as references cited therein.
  • the transgene expression cassette may be a single-stranded AAV (ssAAV) vector or a “dimeric” or self-complementary AAV (scAAV) vector that is packaged as a pseudo-double-stranded transgene.
  • scAAV vectors show an onset of gene expression within hours that plateaus within days after transduction of quiescent cells. Heilbronn.
  • a scAAV is used, where the scAAV has rapid transduction onset and increased stability compared to single stranded AAV.
  • the transgene expression cassette may be split between two AAV vectors, which allows delivery of a longer construct. See e.g., Daya S.
  • a ssAAV vector can be constructed by digesting an appropriate plasmid (such as, for example, a plasmid containing the PGRN gene) with restriction endonucleases to remove the rep and cap fragments, and gel purifying the plasmid backbone containing the AAVwt-ITRs. Choi et al. Subsequently, the desired transgene expression cassette can be inserted between the appropriate restriction sites to construct the single-stranded rAAV vector plasmid.
  • a scAAV vector can be constructed as described in Choi et al.
  • a large-scale plasmid preparation (at least 1 mg) of the rAAV vector and the suitable AAV helper plasmid and pXX6 Ad helper plasmid can be purified by double CsCl gradient fractionation.
  • a suitable AAV helper plasmid may be selected from the pXR series, pXRl-pXR5, which respectively permit cross-packaging of AAV2 ITR genomes into capsids of AAV serotypes 1 to 5.
  • the appropriate capsid may be chosen based on the efficiency of the capsid’s targeting of the cells of interest.
  • transgene expression cassette i.e., transgene expression cassette
  • AAV capsids may be employed to improve expression and/or gene transfer to specific cell types (e.g., retinal cone cells). See, e.g., Yang GS, Virus -mediated transduction of murine retina with adeno-associated virus: Effects of viral capsid and genome size. Journal of Virology, 76(15): 7651-7660.
  • transfection methods for production of AAV may be used in the context of the present invention.
  • transient transfection methods including methods that rely on a calcium phosphate precipitation protocol.
  • the present invention may utilize techniques known in the art for bioreactor-scale manufacturing of AAV vectors, including, for example, Heilbronn; Clement, N. et al. Large-scale adeno-associated viral vector production using a herpesvirus-based system enables manufacturing for clinical studies. Human Gene Therapy, 20: 796-606. [0172] Advances toward achieving the desired goal of scalable production systems that can yield large quantities of clinical grade rAAV vectors have largely been made in production systems that utilize transfection as a means of delivering the genetic elements needed for rAAV production in a cell.
  • adenovirus helper For example, removal of contaminating adenovirus helper has been circumvented by replacing adenovirus infection with plasmid transfection in a three-plasmid transfection system in which a third plasmid comprises nucleic acid sequences encoding adenovirus helper proteins (Xiao, et al. 1998), Improvements in two-plasmid transfection systems have also simplified the production process and increased rAAV vector production efficiency (Grimm, et al. 1998).
  • a second cell-based approach to improving yields of rAAV from cells involves the use of genetically engineered “packaging” cell lines that harbor in their genomes either the AAV rep and cap genes, or both the rep-cap and the ITR-gene of interest (Qiao, el al. 2002).
  • a packaging cell line is either infected or transfected with helper functions, and with the AAV ITR-GOI elements.
  • the latter approach entails infection or transfection of the cells with only the helper functions.
  • rAAV production using a packaging cell line is initiated by infecting the cells with wild -type adenovirus, or recombinant adenovirus. Because the packaging cells comprise the rep and cap genes, it is not necessary to supply these elements exogenously.
  • rAAV yields from packaging cell lines have been shown to be higher than those obtained by proviral cell line rescue or transfection protocols.
  • Amplicon systems are inherently replication-deficient; however the use of a “gutted” vector, replication- competent (rcHSV), or replication-deficient rHSV still introduces immunogenic HSV components into rAAV production systems. Therefore, appropriate assays for these components and corresponding purification protocols for their removal must be implemented.
  • methods for producing recombinant AAV viral particles in a mammalian cell comprising co-infecting a mammalian cell capable of growing in suspension with a first recombinant herpesvirus comprising a nucleic acid sequence encoding an AAV rep and an AAV cap gene each operably linked to a promoter, and a second recombinant herpesvirus comprising a PGRN gene, and a promoter operably linked to said PGRN gene, flanked by AAV inverted terminal repeats to facilitate packaging of the gene of interest, and allowing the virus to infect the mammalian cell, thereby producing recombinant AAV viral particles in a mammalian cell.
  • any type of mammalian cell that is capable of supporting replication of herpesvirus is suitable for use according to the methods of the invention as described herein. Accordingly, the mammalian cell can be considered a host cell for the replication of herpesvirus as described in the methods herein. Any cell type for use as a host cell is contemplated by the present invention, as long as the cell is capable of supporting replication of herpesvirus.
  • suitable genetically unmodified mammalian cells include but are not limited to cell lines such as HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5.
  • the host cells used in the various embodiments of the present invention may be derived, for example, from mammalian cells such as human embryonic kidney cells or primate cells.
  • mammalian cells such as human embryonic kidney cells or primate cells.
  • Other cell types might include, but are not limited to BHK cells, Vero cells, CHO cells or any eukaryotic cells for which tissue culture techniques are established as long as the cells are herpesvirus permissive.
  • the term “herpesvirus permissive” means that the herpesvirus or herpesvirus vector is able to complete the entire intracellular virus life cycle within the cellular environment.
  • methods as described occur in the mammalian cell line BHK, growing in suspension.
  • the host cell may be derived from an existing cell line, e.g., from a BHK cell line, or developed de novo.
  • the methods for producing a rAAV gene construct described herein include also a recombinant AAV viral particle produced in a mammalian cell by the method comprising co-infecting a mammalian cell capable of growing in suspension with a first recombinant herpesvirus comprising a nucleic acid encoding an AAV rep and an AAV cap gene each operably linked to a promoter; and (ii) a second recombinant herpesvirus comprising a PGRN, and a promoter operably linked to said PGRN gene; and allowing the virus to infect the mammalian cell, and thereby producing recombinant AAV viral particles in a mammalian cell.
  • the herpesvirus is a virus selected from the group consisting of: cytomegalovirus (CMV), herpes simplex (HSV) and varicella zoster (VZV) and epstein barr virus (EBV).
  • CMV cytomegalovirus
  • HSV herpes simplex
  • VZV varicella zoster
  • EBV epstein barr virus
  • the recombinant herpesvirus is replication defective.
  • the AAV cap gene has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVrh8, AAVrhlO, AAV2.retro, and variants or hybrids thereof (e.g.
  • AAV2 variants with HSPG mutation (AAV2-HBKO, AAVT-TT, AAV44.9), AAV 1+9 hybrids).
  • the particular capsid sequences confer enhanced neural and brain transduction.
  • the AAV is AAV9.
  • the AAV is AAVrhlO.
  • the first gene cassette is constructed with the gene of interest flanked by inverted terminal repeats (ITRs) from AAV. ITRs function to direct integration of the gene of interest into the host cell genome and are essential for encapsidation of the recombinant genome. Hermonat and Muzyczka, 1984; Samulski et al. 1983.
  • the second gene cassette contains rep and cap, AAV genes encoding proteins needed for replication and packaging of rAAV.
  • the rep gene encodes four proteins (Rep 78, 68, 52 and 40) required for DNA replication.
  • the cap genes encode three structural proteins (VP1, VP2, and VP3) that make up the virus capsid. Muzyczka and Bems, 2001.
  • helper functions are protein products from helper DNA viruses that create a cellular environment conducive to efficient replication and packaging of rAAV.
  • Ad adenovirus
  • herpesviruses can also provide these functions as discussed herein.
  • rAAV vectors for gene therapy is carried out In vitro, using suitable producer cell lines such as BHK cells grown in suspension.
  • suitable producer cell lines such as BHK cells grown in suspension.
  • Other cell lines suitable for use in the invention include HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5.
  • Any cell type can be used as a host cell, as long as the cell is capable of supporting replication of a herpesvirus.
  • One of skill in the art would be familiar with the wide range of host cells that can be used in the production of herpesvirus from host cells.
  • suitable genetically unmodified mammalian host cells may include but are not limited to cell lines such as HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5.
  • a host cell may be adapted for growth in suspension culture.
  • the host cells may be Baby Hamster Kidney (BHK) cells.
  • BHK cell line grown in suspension is derived from an adaptation of the adherent BHK cell line. Both cell lines are available commercially.
  • One strategy for delivering all of the required elements for rAAV production utilizes two plasmids and a helper virus. This method relies on transfection of the producer cells with plasmids containing gene cassettes encoding the necessary gene products, as well as infection of the cells with Ad to provide the helper functions.
  • This system employs plasmids with two different gene cassettes. The first is a proviral plasmid encoding the recombinant DNA to be packaged as rAAV. The second is a plasmid encoding the rep and cap genes, To introduce these various elements into the cells, the cells are infected with Ad as well as transfected with the two plasmids.
  • Ad The gene products provided by Ad are encoded by the genes Ela, Elb, E2a, E4orf6, and Va. Samulski et al. 1998: Hauswirth et al. 2000; Muzyczka and Bums, 2001.
  • the Ad infection step can be replaced by transfection with an adenovirus “helper plasmid” containing the VA, E2A and E4 genes. Xiao et al. 1998; Matsushita, et al. 1998.
  • HSV-1 herpes simplex vims type 1
  • the minimal set of HSV-1 genes required for AAV2 replication and packaging has been identified, and includes the early genes UL5, UL8, UL52 and UL29. Muzyczka and Bums, 2001. These genes encode components of the HSV-1 core replication machinery, i.e., the helicase, primase, primase accessory proteins, and the single-stranded DNA binding protein. Knipe, 1989; Weller, 1991.
  • This rAAV helper property of HSV-1 has been utilized in the design and construction of a recombinant herpes vims vector capable of providing helper vims gene products needed for rAAV production. Conway et al. 1999.
  • rAAV vectors for gene therapy is carried out In vitro, using suitable producer cell lines such as BHK cells grown in suspension.
  • suitable producer cell lines such as BHK cells grown in suspension.
  • Other cell lines suitable for use in the invention include HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5.
  • Any cell type can be used as a host cell, as long as the cell is capable of supporting replication of a herpesvirus.
  • One of skill in the art would be familiar with the wide range of host-cells that can be used in the production of herpesvirus from host cells.
  • suitable genetically unmodified mammalian host cells may include but are not limited to cell lines such as HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5.
  • a host cell may be adapted for growth in suspension culture.
  • the host cells are Baby Hamster Kidney (BHK) cells.
  • BHK cell line grown in suspension is derived from an adaptation of the adherent BHK cell line. Both cell lines are available commercially.
  • stirred tank bioreactors provide very high volume-specific culture surface area and has been used for the production of viral vaccines (Griffiths, 1986). Furthermore, stirred tank bioreactors have industrially been proven to be scalable. One example is the multiplate CELL CUBE cell culture system. The ability to produce infectious viral vectors is increasingly important to the pharmaceutical industry, especially in the context of gene therapy.
  • Bioreactors have been widely used for the production of biological products from both suspension and anchorage dependent animal cell cultures. Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up. However, continuous processes based on chemostat or perfusion principles are available.
  • the bioreactor system may be set up to include a system to allow for media exchange. For example, filters may be incorporated into the bioreactor system to allow for separation of cells from spent media to facilitate media exchange.
  • media exchange and perfusion is conducted beginning on a certain day of cell growth.
  • media exchange and perfusion can begin on day 3 of cell growth.
  • the filter may be external to the bioreactor, or internal to the bioreactor.
  • a method for producing recombinant AAV viral particles may comprise: co-infecting a suspension cell with a first recombinant herpesvirus comprising a nucleic acid encoding an AAV rep and an AAV cap gene each operably linked to a promoter; and a second recombinant herpesvirus comprising a PGRN gene construct, and a promoter operably linked to said gene of interest; and allowing the cell to produce the recombinant AAV viral particles, thereby producing the recombinant AAV viral particles.
  • the cell may be HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5.
  • the cap gene may be selected from an AAV with a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrhlO, AAV2. retro, and variants or hybrids thereof (e.g. AAV2 variants with HSPG mutation, (AAV2-HBKO, AAVT-TT, AAV44.9), AAV 1+9 hybrids).
  • the particular capsid sequences confer enhanced neural and brain transduction.
  • the AAV is AAV9.
  • the AAV is AAVrhlO.
  • the cell may be infected at a combined multiplicity of infection (MOI) of between 3 and 14.
  • the first herpesvirus and the second herpesvirus may be viruses selected from the group consisting of: cytomegalovirus (CMV), herpes simplex (HSV) and varicella zoster (VZV) and Epstein Barr Virus (EBV).
  • CMV cytomegalovirus
  • HSV herpes simplex
  • VZV varicella zoster
  • EBV Epstein Barr Virus
  • the herpesvirus may be replication defective.
  • the co-infection may be simultaneous.
  • the recombinant AAV viral particles further comprise a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • a method for producing recombinant AAV viral particles in a mammalian cell may comprise co infecting a suspension cell with a first recombinant herpesvirus comprising a nucleic acid encoding an AAV rep and an AAV cap gene each operably linked to a promoter; and a second recombinant herpesvirus comprising a PGRN gene construct, and a promoter operably linked to said PGRN gene construct; and allowing the cell to propagate, thereby producing the recombinant AAV viral particles, whereby the number of viral particles produced is equal to or greater than the number of viral particles grown in an equal number of cells under adherent conditions.
  • the cell may be HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5.
  • the cap gene may be selected from an AAV with a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVrh8, AAVrhlO, AAV2.retro, and variants or hybrids thereof (e.g. AAV2 variants with HSPG mutation, (AAV2-HBKO, AAVT-TT, AAV44.9), AAV 1+9 hybrids).
  • the AAV is AAVrhlO.
  • the AAV is AAV9.
  • the particular capsid sequences confer enhanced neural and brain transduction.
  • the cell may be infected at a combined multiplicity of infection (MOI) of between 3 and 14.
  • the first herpesvirus and the second herpesvirus may be viruses selected from the group consisting of: cytomegalovirus (CMV), herpes simplex (HSV) and varicella zoster (VZV) and Epstein Barr Virus (EBV).
  • the herpesvirus may be replication defective.
  • the co-infection may be simultaneous.
  • a method for delivering a nucleic acid sequence encoding a therapeutic protein to a suspension cell comprising: co-infecting the BHK cell with a first recombinant herpesvirus comprising a nucleic acid encoding an AAV rep and an AAV cap gene each operably linked to a promoter; and a second herpesvirus comprising a PGRN gene construct, wherein the gene of interest comprises a therapeutic protein coding sequence, and a promoter operably linked to said PGRN gene; and wherein said cell is infected at a combined multiplicity of infection (MOI) of between 3 and 14; and allowing the virus to infect the cell and express the therapeutic protein, thereby delivering the nucleic acid sequence encoding the therapeutic protein to the cell.
  • MOI multiplicity of infection
  • the cell may be HEK-293 (293), Vero, RD, BHK-21, HT- 1080, A549, Cos-7, ARPE-19, and MRC-5. See, e.g., U.S. Patent No. 9,783,826. V. Meth o ds of T reatment
  • Gene therapy refers to treatment of inherited or acquired diseases by replacing, altering, or supplementing a gene responsible for the disease. It is achieved by introduction of a corrective gene or genes into a host cell, generally by means of a vehicle or vector.
  • Gene therapy using rAAV holds great promise for the treatment of many diseases.
  • a method of producing recombinant adeno-associated virus (rAAV), and in particular producing large quantities of recombinant AAV, to support treatment of neurodegenerative diseases are described herein.
  • rAAV recombinant AAV
  • rAAV has been used successfully as a gene therapy vehicle to enable expression of erythropoietin in skeletal muscle of mice (Kessler, et al. 1996), tyrosine hydroxylase and aromatic amino acid decarboxylase in the CNS in monkey models of Parkinson disease (Kaplitt, et al. 1994) and Factor IX in skeletal muscle and liver in animal models of hemophilia.
  • the rAAV vector has been used in human clinical trials to deliver the CFTR gene to cystic fibrosis patients and the Factor IX gene to hemophilia patients (Flotte, et al. 1998; Wagner, et al. 1998), Further, AAV is a helper-dependent DNA parvovirus, which is not associated with disease in humans or mammals (Bems and Bohensky, 1987, Advances in Virus Research, Academic Press Inc, 32:243-307). Accordingly, one of the most important attributes of AAV vectors is their safety profile in phase I clinical trials.
  • AAV gene therapy has been carried out in a number of different pathological settings and to treat a various diseases and disorders.
  • administration of an AAV2-FIX vector into the skeletal muscle of eight hemophilia B subjects proved safe and achieved local gene transfer and Factor IX expression for at least 10 months after vector injection (Jiang, et al. Mol Ther. 14 (3):452-5 2006)
  • rAAV2-CB-hAAT a recombinant adeno-associated virus alpha 1- antitrypsin
  • V-GAD gene therapy of the subthalamic nucleus has been shown to be safe and well tolerated by patients with advanced Parkinson’s disease (Kaplitt et al. Lancet. 200723; 369(9579):2097-105).
  • the neurodegenerative disease is mediated by a heritable mutation in the subject.
  • the neurodegenerative disease is mediated by an environmental insult to the subject.
  • a neurodegenerative disease mediated by an environmental insult to the patient means a disease that is caused by an environmental insult and is not caused by a heritable mutation of the progranulin gene that modifies progranulin expression.
  • a heritable mutation is a permanent mutation in a patient's DNA that may be transmitted to the patient's offspring. Delivery of one or more of the nucleic acids described herein to CNS cells, and in particular to neuronal cells, can be used to treat neurodegenerative diseases.
  • methods are provided herein that employ PGRN AAV-based gene therapy for treating a progranulin-associated neurodegenerative disease including but not limited to familial frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), neuronal ceroid lipofuscinosis (NCL), including neuronal ceroid lipofuscinosis-11 (CLN11) and Batten disease, and Alzheimer’s disease (AD).
  • FDD familial frontotemporal dementia
  • FTLD frontotemporal lobar degeneration
  • NCL neuronal ceroid lipofuscinosis
  • CLN11 neuronal ceroid lipofuscinosis-11
  • Batten disease Alzheimer’s disease
  • methods are provided herein that employ PGRN AAV-based gene therapy for preventing progranulin-associated neurodegenerative disorder including but not limited to familial frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), neuronal ceroid lipofuscinosis (NCL), including neuronal ceroid lipofuscinosis-11 (CLN11) and Batten disease, and Alzheimer’s disease (AD).
  • FDD familial frontotemporal dementia
  • FTLD frontotemporal lobar degeneration
  • NCL neuronal ceroid lipofuscinosis
  • CLN11 neuronal ceroid lipofuscinosis-11
  • Batten disease Alzheimer’s disease
  • the methods described herein allow for the production of recombinant AAV viral particles in a mammalian cell comprising co-infecting a mammalian cell capable of growing in suspension with a first recombinant herpesvirus and a second recombinant herpesvirus comprising a progranulin gene construct that has therapeutic value in the treatment of progranulin-associated neurodegenerative disorder including but not limited to familial frontotemporal dementia (FTD) and neuronal ceroid lipofuscinosis- 11 (CLN11).
  • FDD familial frontotemporal dementia
  • CLN11 neuronal ceroid lipofuscinosis- 11
  • Progranulin-associated neurodegenerative disorders include, but are not limited to, 20% of the incidences of familial frontotemporal dementia (FTD) and all instances of neuronal ceroid lipofuscinosis- 11 (CLN11).
  • FDD familial frontotemporal dementia
  • FTLD frontotemporal lobar degeneration
  • NCL neuronal ceroid lipofuscinosis
  • AD Alzheimer’s disease
  • Progranulin a secreted glycoprotein
  • Progranulin is encoded in humans by the single GRN gene.
  • Progranulin (PGRN) is predominantly expressed by microglia in the brain.
  • Progranulin (PGRN) is a secreted 593 amino acid multifunction protein that is highly conserved and found in a wide range of species ranging from eukaryotes to humans. PGRN is widely distributed throughout the CNS where it is found primarily in neurons and microglia but has also been detected, at much lower levels, in astrocytes and oligodendrocytes.
  • Progranulin consists of seven and a half, tandemly repeated, non-identical copies of the 12 cysteine granulin motif.
  • Frontotemporal dementia is an early onset form of dementia, distinct from Alzheimer’s disease.
  • the GRN-related form of frontotemporal lobar dementia is a proteinopathy characterized by the appearance of neuronal inclusions containing ubiquitinated and fragmented TDP-43 (encoded by TARDBP). Chitramuthu el al. Brain 2017 140(12): 3081-3104; Suarez-Calvet et al. EMBO Molecular Medicine 2018 e9712.
  • the progranulin (PGRN) AAV construct described herein provides a gene therapy vehicle for the treatment of PGRN-associated neurodegenerative disorders, including 20% of all incidences of familial frontotemporal dementia (FTD) and all instances of neuronal ceroid lipofucinosis-11 (CLN11).
  • FTD familial frontotemporal dementia
  • CLN11 neuronal ceroid lipofucinosis-11
  • the PGRN AAV gene therapy construct and methods of use described herein provides a therapy for PGRN- associated neurodegenerative disorders, a long-felt unmet need as there are no gene therapy -based treatments available for patients suffering from PGRN-associated neurodegenerative disorders.
  • the PGRN AAV gene therapy is administered before the subject has developed a neurodegenerative disease.
  • the subject is diagnosed with a neurodegenerative disease by molecular genetic testing to identify PGRN mutation.
  • the subject has a family member with a neurodegenerative disease.
  • the rAAV constructs described herein transduce CNS cells, and in particular neuronal cells, with greater efficiency than do conventional AAV vectors.
  • the compositions and methods described herein enable the highly efficient delivery of nucleic acids to CNS cells, and in particular neuronal cells.
  • compositions and methods described herein enable the delivery to, and expression of, a transgene in at least 50%, (e.g., at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of inner hair cells or delivery to, and expression in, at least 50%, (e.g., at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99) of neuronal cells.
  • a transgene in at least 50%, (e.g., at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99) of neuronal cells.
  • compositions and methods described herein enable the delivery to, and expression of, a transgene in at least 70%, (e.g., at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of inner hair cells or delivery to, and expression in, at least 70%, (e.g., at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99) of neuronal cells.
  • a transgene in at least 70%, (e.g., at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99) of neuronal cells.
  • compositions and methods described herein enable the delivery to, and expression of, a transgene in at least 80%, (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of inner hair cells or delivery to, and expression in, at least 80%, (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99) of neuronal cells.
  • the nucleic acid sequences described herein are directly introduced into a cell, where the nucleic acid sequences are expressed to produce the encoded product, prior to administration in vivo of the resulting recombinant cell. This can be accomplished by any of numerous methods known in the art, e.g., by such methods as electroporation, lipofection, calcium phosphate mediated transfection.
  • compositions comprising any of the vectors described herein, optionally in a pharmaceutically acceptable excipient.
  • excipients are relatively inert substances that facilitate administration of a pharmacologically effective substance and can be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in liquid prior to use.
  • an excipient can give form or consistency, or act as a diluent.
  • Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, pH buffering substances, and buffers.
  • excipients include any pharmaceutical agent suitable for direct delivery to the ear (e.g., inner ear or middle ear) which may be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of the various TWEEN compounds, and liquids such as water, saline, glycerol and ethanol.
  • Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present.
  • Methods for reducing aggregation of rAAVs are well known in the art and, include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright FR, el al, Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference.)
  • the pharmaceutical composition comprises one or more of BSST, PBS or BSS.
  • the pharmaceutical composition further comprises histidine buffer.
  • compositions may optionally be supplied in unit dosage form suitable for administration of a precise amount.
  • compositions described herein are formulated for administration to the CNS.
  • the compositions are formulated for administration to neuronal cells.
  • administration is intracerebral (e.g. to the intra-cistema magna (ICM)), intrathecal (IT), with or without catheter, intravenous (IV), or a combination of IV and IT.
  • ICM intra-cistema magna
  • IV intravenous
  • IT intravenous
  • intrathecal administration refers to the administration of an agent, e.g., a composition comprising a rAAV, into the spinal canal.
  • an agent e.g., a composition comprising a rAAV
  • intrathecal administration may comprise injection in the cervical region of the spinal canal, in the thoracic region of the spinal canal, or in the lumbar region of the spinal canal.
  • intrathecal administration is performed by injecting an agent, e.g., a composition comprising a rAAV, into the subarachnoid cavity (subarachnoid space) of the spinal canal, which is the region between the arachnoid membrane and pia mater of the spinal canal.
  • intrathecal administration is not administration into the spinal vasculature.
  • Intracerebral administration refers to administration of an agent into and/or around the brain.
  • Intracerebral administration includes, but is not limited to, administration of an agent into the cerebrum, medulla, pons, cerebellum, intracranial cavity, and meninges surrounding the brain.
  • Intracerebral administration may include administration into the dura mater, arachnoid mater, and pia mater of the brain.
  • Intracerebral administration may include, in some embodiments, administration of an agent into the cerebellomedullary (CM) cistern.
  • CM cerebellomedullary
  • Intracerebral administration may include, in some embodiments, administration of an agent into the cerebrospinal fluid (CSF) of the subarachnoid space surrounding the brain.
  • Intracerebral administration may include, in some embodiments, administration of an agent into ventricles of the brain, e.g., the right lateral ventricle, the left lateral ventricle, the third ventricle, the fourth ventricle.
  • intracerebral administration is not administration into the brain vasculature.
  • Intracerebral administration may involve direct injection into and/or around the brain.
  • intracerebral administration involves injection using stereotaxic procedures.
  • Stereotaxic procedures are well known in the art and typically involve the use of a computer and a 3- dimensional scanning device that are used together to guide injection to a particular intracerebral region, e.g., a ventricular region.
  • Micro -injection pumps e.g., from World Precision Instruments
  • a microinjection pump is used to deliver a composition comprising a rAAV.
  • the infusion rate of the composition is 1 mL/minute or slower. According to some embodiments, the infusion rate of the composition is between about 10m1 / minute to 1000 m ⁇ / minute. As will be appreciated by the skilled artisan, infusion rates will depend on a variety of factors, including, for example, species of the subject, age of the subject, weight/size of the subject, serotype of the AAV, dosage required, intracerebral region targeted, etc. Thus, other infusion rates may be deemed by a skilled artisan to be appropriate in certain circumstances.
  • the composition comprising an rAAV as described herein is administered using an osmotic pump or an infusion pump.
  • osmotic and infusion pumps are commercially available from a variety of suppliers, for example Alzet Corporation, Hamilton Corporation, Alza, Inc., Palo Alto, Calif.).
  • the methods of the invention may be used to treat an individual e.g., a human, wherein the transduced cells produce PGRN in an amount sufficient to treat or prevent a neurodegenerative disease.
  • the volume of vector delivered may be determined based on the characteristics of the subject receiving the treatment, such as the age of the subject and the volume of the area to which the vector is to be delivered.
  • the volume of the composition injected is between about 10 m ⁇ to about 1000 m ⁇ , or between about between about 100 m ⁇ to about 1000 m ⁇ , or between about between about 100 m ⁇ to about 500 m ⁇ , or between about 500 m ⁇ to about 1000 m ⁇ .
  • the volume of the composition injected is more than about any one of 1 m ⁇ , 2 m ⁇ , 3 m ⁇ , 4 m ⁇ , 5 m ⁇ , 6 m ⁇ , 7 m ⁇ , 8 m ⁇ , 9 m ⁇ , 10 m ⁇ , 15 m ⁇ , 20 m ⁇ , 25 m ⁇ , 50 m ⁇ , 75 m ⁇ , 100 m ⁇ , 200 m ⁇ , 300 m ⁇ , 400 m ⁇ , 500 m ⁇ , 600 m ⁇ , 700 m ⁇ , 800 m ⁇ , 900 m ⁇ , or 1 mL, or any amount there between.
  • the concentration of vector that is administered may differ depending on production method and may be chosen or optimized based on concentrations determined to be therapeutically effective for the particular route of administration.
  • the concentration in vector genomes per milliliter (vg/ml) is selected from the group consisting of about 10 8 vg/ml, about 10 9 vg/ml, about 10 10 vg/ml, about 10 11 vg/ml, about 10 12 vg/ml, about 10 13 vg/ml, and about 10 14 vg/ml.
  • the concentration is in the range of 10 11 vg/ml - 10 14 vg/ml in a volume of about 0.1 mL, about 0.2 mL, about 0.4 mL, about 0.6 mL, about 0.8 mL, and about 1.0 mL.
  • the effectiveness of the compositions described herein can be monitored by several criteria.
  • effectiveness of the compositions is determined by monitoring an improvement in subjects treated with PGRN rAAV.
  • the effectiveness of the compositions described herein can be monitored in an in vivo mouse model. For example, in a PGRN-/-KO mouse model, increased PGRN protein levels in blood, CSF and brain tissue; decreased levels of neurofilament-1 (Nfl-1) in blood and CSF; decreased levels of lipofuscin and intracellular TDP43 from brain tissue may be indicators of the effectiveness of the compositions.
  • the effectiveness of the compositions described herein can be monitored in human disease subjects. For example, increased PGRN protein levels in blood and CSF, decreased levels of Nfl-1 in blood and CSF, and behavioral and cognitive improvements may be used as indicators of the effectiveness of the compositions.
  • the rHSV co-infection method for recombinant adeno-associated virus (rAAV) production employs two ICP27-deficient recombinant herpes simplex virus type 1 (rHSV-1) vectors, one bearing the AAV rep and cap genes (rHSV-rep2capX, with “capX” referring to any of the AAV serotypes), and the second bearing the gene of interest (GO I) cassette flanked by AAV inverted terminal repeats (ITRs).
  • the system was developed with AAV serotype 2 rep, cap, and ITRs, as well as the humanized green fluorescent protein gene (GFP) as the transgene, the system can be employed with different transgenes and serotype/pseudo type elements.
  • GFP humanized green fluorescent protein gene
  • Mammalian cells are infected with the rHSV vectors, providing all cis and trans-acting rAAV components as well as the requisite helper functions for productive rAAV infection.
  • Cells are infected with a mixture of rHSV-rep2capX and rHSV-GOI.
  • Cells are harvested and lysed to liberate rAAV-GOI, and the resulting vector stock is titered by the various methods described below.
  • An alternative method for harvesting rAAV is by in situ lysis. At the time of harvest, MgCb is added to a final concentration of 1 mM, 10% (v/v) Triton X-100 added to a final concentration of 1% (v/v), and Benzonase is added to a final concentration of 50 units/mL. This mixture is either shaken or stirred at 37°C for 2 hours. Quantitative real-time PCR to determine DRP yield
  • the DNAse-resistant particle (DRP) assay employs sequence-specific oligonucleotide primers and a dual-labeled hybridizing probe for detection and quantification of the amplified DNA sequence using real-time quantitative polymerase chain reaction (qPCR) technology.
  • the target sequence is amplified in the presence of a fluorogenic probe which hybridizes to the DNA and emits a copy- dependent fluorescence.
  • the DRP titer (DRP/mL) is calculated by direct comparison of relative fluorescence units (RFUs) of the test article to the fluorescent signal generated from known plasmid dilutions bearing the same DNA sequence.
  • the data generated from this assay reflect the quantity of packaged viral DNA sequences, and are not indicative of sequence integrity or particle infectivity.
  • Green-cell infectivity assay to determine infectious particle yield rAA V-GFP only
  • Infectious particle (ip) titering is performed on stocks of rAA V-GFP using a green cell assay.
  • C12 cells (a HeLa derived line that expressed AAV2 Rep and Cap genes - see references below) are infected with serial dilutions of rAA V-GFP plus saturating concentrations of adenovirus (to provide helper functions for AAV replication). After two to three days incubation, the number of fluorescing green cells (each cell representing one infectious event) are counted and used to calculate the ip/mL titer of the virus sample.
  • Clark KR et al. described recombinant adenoviral production in Hum. Gene Ther. 1995. 6:1329- 1341 and Gene Ther. 1996. 3:1124-1132, both of which are incorporated by reference in their entireties herein.
  • rAAV-GOI tissue culture infectious dose at 50% (TCID50) assay. Eight replicates of rAAV were serially diluted in the presence of human adenovirus type 5 and used to infect HeLaRC32 cells (a HeLa-derived cell line that expresses AAV2 rep and cap, purchased from ATCC) in a 96-well plate.
  • lysis buffer final concentrations of 1 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.25% (w/v) deoxycholate, 0.45% (v/v) Tween-20, 0.1% (w/v) sodium dodecyl sulfate, 0.3 mg/mL Proteinase K
  • lysis buffer final concentrations of 1 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.25% (w/v) deoxycholate, 0.45% (v/v) Tween-20, 0.1% (w/v) sodium dodecyl sulfate, 0.3 mg/mL Proteinase K
  • rAAV vectors for gene therapy is carried out in vitro, using suitable producer cell lines such as HEK293 cells (293).
  • suitable producer cell lines such as HEK293 cells (293).
  • Other cell lines suitable for use in the invention include Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5.
  • DMEM Dulbecco’s modified Eagle’s medium
  • FBS fetal bovine serum
  • Cells can be grown to various concentrations including, but not limited to at least about, at most about, or about 1 x 10 6 to 4 x 10 6 cells/mL. The cells can then be infected with recombinant herpesvirus at a predetermined MOI.
  • AAV-Progranulin therapy for neurodegenerative disorders is critically dependent on the vector component.
  • An optimal vector will provide an efficient capsid that targets the brain effectively, a moderate but cell-specific promoter and a stabilized transgene expressing human progranulin protein.
  • FIG. 1 shows a schematic of a PGRN construct, comprising inverted terminal repeats (ITR) at each end, a human synapsin 1 (hSYNl) or chicken beta actin (CBA) promoter, PGRN optionally comprising a C-terminal HA tag or a deletion of 3-16 nucleic acids in the C-terminal, Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE), SV40 early polyadenylation signals (SV40pA) and optionally staffer DNA.
  • the bottom panel of FIG. 1A shows a self complementary AAV -genome.
  • CBA promoter was obtained in a pAAV plasmid (pTR-CBA-PGRNwt-WPRE-pA). The sequence of PGRNwt was replaced with a sequence containing hGFP and a multiple cloning site using NEBuilder® HiFi DNA Assembly kit (New England Biolabs) to generate the plasmid pTR-CBA-hGFP-WPRE-pA.
  • the human synapsin promoter sequence was synthesized commercially (Genscript), followed by in-house PCR amplification and extraction, resulting in a promoter segment with compatible restriction site segments for insertion into a unique viral packaging vector containing an ampicillin selection cassette, AAV ITR segments, hGFP reporter gene, WPRE and SV40 poly A (pTR-hSyn-hGFP-WPRE-pA).
  • AAV9 and AAVrhlO both possess broad neuronal tropism favorable for therapy.
  • AAVrhlO was chosen over AAV9 based on studies comparing AAV9 and AAVrhlO expression and efficacy in the central nervous system.
  • AAVrhlO was found to transduce significantly better than AAV9.
  • AAV9 and rhlO have similarly low neutralizing Ab seroprevalences in the human population (18% and 21% respectively, Thwaite R et al. 2014).
  • FIG. 25 summarizes the results for each of the capsids tested, for percent GFP positive cells and GFP intensity in various regions of the brain after intrathecal injection. From this study, AAVrhlO showed the most GFP expression, followed by AAV9. All of the vectors were well-tolerated.
  • AAVrhlO biodistribution in NHP by dosing into the cistema magna (ICM) was carried out. It was found that 1.2E13 vg of AAVRhlO showed excellent biodistribution from frontal to dorsal NHP brain following ICM dosing. Scoring is shown in FIG. 26.
  • hSyn promoter also called SYNP1, neuron specific
  • CBA chimeric CMV-chicken b- actin promoter
  • FIG. 28 shows GFP expression after rAAVRhlO-CBA-hGFP Transduction in HEK293 and SHSY-5Y cells, with and without AD5 vector.
  • SH-SY5Y cells transduced with the AAVRhlO-CBA construct, with and without AD5
  • no GFP was detected.
  • the results shown in FIG. 27 demonstrate that both HEK and SH-SY5Y cells can be used to showcase WPRE-enhanced expression driven from either CBA or hSYN promoter.
  • the level of hSYN-driven expression is considerably lower than CBA-driven expression in both cell types.
  • AAV2tYF or AAVrhlO can be used to showcase transduction of vector in HEK cells (and is further enhanced by Ad5), but only AAV2tYF can be used to showcase transduction in SH-SY5Y cells (regardless of Ad5 addition).
  • Progranulin transgene optimization consists of efforts to enhance protein expression, stability, and function. Codon optimized variants of Progranulin (PGRN) were synthesized and assessed for changes in protein expression versus wildtype. Six codon optimized variants were generated, either with or without 27 bp C-terminal HA tag. The HA-tagged variants provide an alternative measure for protein expression, a means for discriminating over endogenous PGRN protein, and also allow the assessment of any therapeutic benefit to inhibiting PGRN-Sortilin binding.
  • PGRN Progranulin
  • Each codon optimized variant contains unique optimizations (i.e., codon usage, GC content, stability of 5’ mRNA structure, removal of RNA destabilizing sequences, etc.), that led to variants which vary in their DNA sequences but preserve their amino acid sequences. These variants were generated from one of three different optimization algorithms (or a combination thereof).
  • PCR amplification and extraction was performed, resulting in PGRN transgene segments with compatible restriction sites (Notl, Nhel) for insertion into a unique viral packaging vector containing an ampicillin selection cassette and AAV ITR segments.
  • codon optimized constructs were transformed into high efficiency E. coli cells (SURE2) for amplification, and clones were selected for validation.
  • Sanger sequencing (Genewiz) and restriction digests were performed to validate the codon optimized PGRN and PGRNwt plasmids. Positive clones for each codon optimized construct were selected for subsequent experiments.
  • coE Codon optimized variants coE
  • coF coF
  • PGRNcoE and coF PGRNcoF
  • hSY5Y cells were transfected into SH-SY5Y cells and secreted transgene expression was analyzed via supernatant ELISA.
  • coE showed comparable secreted progranulin expression (ng/ml) to WT, and higher secreted progranulin expression (ng/ml) than coF in HEK293 cells. Expression was also measured at day 5 (d5), where a slight decrease in secreted progranulin expression was seen with the variant coE, while an increase was seen with the variant coF.
  • rAAV-X vectors were produced, where “X” is one of the following serotypes/variants: AAV- rhlO, AAV-9 containing top selected genomic variants. Research grade preparation of these vectors will proceed by various methods.
  • vector is produced using AGTC’s proprietary recombinant HSV complementation in suspension-cultured baby hamster kidney (sBHK) cells (Kang et ak, Gene Ther 2009; Thomas et ak, Hum Gene Ther 2009).
  • a triple transfection method was used to make vector for all preclinical work. Triple transfection will also be used to make vector for GLP tox studies. Vector production is being optimized using the HAVE (HSV-associated) method and the method is expected to be used for production in later stage work, such as for preparation of clinical trial material. Studies will be carried out to demonstrate comparability of vector made by both methods.
  • HAVE HSV-associated
  • a vector construct comprising rAAVrlO-SYN-PGRNwt was prepared by triple transfection, comprising the pUC-based “pTR” plasmid with the ITR-SYN-PGRNwt- wpre-pA-ITR cassette inserted.
  • the nucleic acid sequence of the ITR-SYN-PGRNwt-wpre-pA-ITR cassette can be derived from individual sequences provided in FIG. 4 or FIG. 5A and 5B (ITR sequences) or 5AB (for both ITRs), FIG. 3 (hSYN), FIG. 6 (hPGRNwt), FIG. 17 (for WPRE), and FIG. 18 (for SV40pA).
  • Example 6 Progranulin Expression in Non-Human Primates
  • a study was conducted to evaluate systemic gene expression following a single intracistemal injection of a gene therapy (using rAAVrlO-SYN-PGRNwt ) in the cynomolgus monkey.
  • a gene therapy using rAAVrlO-SYN-PGRNwt
  • two male cynomolgus monkeys one of which was previously implanted with an intrathecal lumbar catheter for CSF sample collection (Animal 002A) were transferred to the study.
  • the animals were administered a single dose of 1.5 mL of test article by cistema magna puncture according to the following study design.
  • CM Access to the CM was confirmed by the flow of CSF from the needle. Once CSF was observed in the hub of the spinal needle, the 1.5 mL dose was administered over approximately 1-2 minutes by manual bolus. Upon removing the needle, direct pressure was applied to the injection site followed by application of a topical aseptic ointment. The animals were placed in the Trendelenburg position for 10 minutes following dose administration, prior to anesthetic reversal. The reversal agent, atipamezole hydrochloride, was provided at a dose of 0.2 mg/kg IM, the time recorded and the animal returned to its cage. [0273] In-life observations and measurements included clinical observations, body weight, and clinical pathology evaluations as described elsewhere in this report. Blood and CSF were collected for analysis. After collection of samples on Day 57, both animals were returned to the NBR primate stock colony. [0274] There were no abnormal clinical signs or test article-related changes in body weight over the course of the study.
  • Non-Patent Literature All publications (e.g., Non-Patent Literature), patents, patent application publications, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All such publications (e.g., Non-Patent Literature), patents, patent application publications, and patent applications are herein incorporated by reference to the same extent as if each individual publication, patent, patent application publication, or patent application was specifically and individually indicated to be incorporated by reference.

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AU2020371662A AU2020371662A1 (en) 2019-10-22 2020-10-22 Adeno-associated virus (AAV) systems for treatment of progranulin associated neurodegenerative diseases or disorders
KR1020227017064A KR20230019402A (ko) 2019-10-22 2020-10-22 프로그래뉼린 연관 신경변성 질환 또는 장애의 치료를 위한 아데노-연관 바이러스 (aav) 시스템
CA3158516A CA3158516A1 (en) 2019-10-22 2020-10-22 Adeno-associated virus (aav) systems for treatment of progranulin associated neurodegeneative diseases or disorders
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EP20880235.5A EP4048799A4 (en) 2019-10-22 2020-10-22 ADENO-ASSOCIATED VIRUS (AAV) SYSTEMS FOR THE TREATMENT OF PROGRANULIN-ASSOCIATED NEURODEGENERATIVE DISEASES OR DISORDERS
CN202080089386.1A CN115715327A (zh) 2019-10-22 2020-10-22 用于治疗颗粒蛋白前体相关的神经退行性疾病或病症的腺伴随病毒(aav)系统
JP2022523408A JP2022553307A (ja) 2019-10-22 2020-10-22 プログラニュリン関連神経変性疾患または障害の治療のためのアデノ随伴ウイルス（ａａｖ）システム
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