WO2024220545A2 - Capsides d'aav pour administration sur cible à la moelle épinière - Google Patents

Capsides d'aav pour administration sur cible à la moelle épinière Download PDF

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WO2024220545A2
WO2024220545A2 PCT/US2024/024996 US2024024996W WO2024220545A2 WO 2024220545 A2 WO2024220545 A2 WO 2024220545A2 US 2024024996 W US2024024996 W US 2024024996W WO 2024220545 A2 WO2024220545 A2 WO 2024220545A2
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aav
spinal cord
aav9
capsids
seq
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WO2024220545A3 (fr
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Casey A. MAGUIRE
Killian S. HANLON
Demitri Alexander DE LA CRUZ
Mingjie CHENG
Miguel Israel Chavez SANTOSCOY
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General Hospital Corp
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General Hospital Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • AAV capsids with improved biodistribution and transduction efficiency in the spinal cord and lowered biodistribution to liver, compositions comprising the capsids, and methods of using the same.
  • AAV adeno-associated virus
  • Systemic dosing also has major translational challenges such as pre-existing neutralizing antibodies to the AAV capsid, the high cost of generating the massive doses (>10 14 vg/kg) required for therapy, and even dose limiting toxicities due to apparent complement activation and liver dysfunction 4, 5 .
  • DRG dorsal root ganglion
  • Described herein are engineered capsids, including AAV capsid proteins comprising an amino acid sequence that comprises at least four, at least five, at least six, or all seven contiguous amino acids from the sequence TR2 (RTTASLM, SEQ ID NO: 12), NL1 (LTTEGRR, SEQ ID NO: 10), TH1 (HPARALP, SEQ ID NO: 13), or TP1 (PKYPLLG, SEQ ID NO: 14), or another amino acid sequence described herein, e.g., in any of Tables III, IV, V, VIA, VIB, or VIC.
  • the AAV is AAV9, e.g., AAV9 VP1.
  • the sequence is inserted in a position corresponding to amino acids 588 and 589 of AAV9 VP1.
  • nucleic acids encoding the AAV capsid proteins described herein, optionally as recombinant episomes or viral vectors.
  • AAV comprising a capsid protein as described herein, and preferably not comprising a wild type VP1, VP2, or VP3 capsid protein.
  • the AAV further comprises a transgene, preferably a therapeutic transgene, e.g., a protein coding sequence or inhibitory nucleic acid as listed in Table A.
  • a transgene preferably a therapeutic transgene, e.g., a protein coding sequence or inhibitory nucleic acid as listed in Table A.
  • the methods comprise contacting the cell with an AAV comprising a capsid protein as described herein.
  • the cell is a neuron (optionally a dorsal root ganglion neuron or spiral ganglion neuron), astrocyte, glial cell, Schwann cell of a peripheral nerve, or cardiomyocyte.
  • the cell is in a living subject, e.g., a mammalian subject.
  • the cell is in a tissue selected from the brain, spinal cord, dorsal root ganglion, Schwann cell of a peripheral nerve, or heart, and a combination thereof.
  • the subject has a disease listed in Table A, and the methods include delivering an AAV comprising a capsid protein as described herein and a protein coding sequence or inhibitory nucleic acid as listed in Table A.
  • the cell is in the spinal cord of the subject, and the AAV is administered by intrathecal delivery.
  • the intrathecal delivery is via lumbar injection, cisternal magna injection, or intraparenchymal injection.
  • the AAV is delivered by parenteral delivery, preferably via intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular delivery.
  • FIGs. 1A-B Overview of the in vivo selection in non-human primates (NHPs) to identify AAV capsids with the ability to efficiently transduce spinal cord.
  • the iTransduce AAV genome plasmid has a CBA- driven Cre cassette followed by a p41 driven AAV9 capsid with randomized 21mer bp inserts encoding for 7mer peptides inserted into the capsid.
  • NHPs are injected intrathecally (IT) with the library and three weeks later animals are sacrificed and spinal cord removed.
  • Next generation sequencing (NGS) is performed on AAV genomes containing 21mer inserts amplified from three spinal cord regions.
  • the rescued cap inserts are inserted into the iTransduce backbone and packaged into the R1 rescued library, b. Round 2 of selection.
  • a second reporter AAV expression plasmid is used to allow transduction-based selection in a non-transgenic animal.
  • the reporter contains a Cre-sensitive floxed-STOP-H2B-mPlum cassette.
  • Cre-sensitive floxed-STOP-H2B-mPlum cassette When coinjected intrathecally into NHPs with iTransduce library from Round 1, cells that are co-transduced with Cre-expressing capsids can rescue nuclear mPlum expression.
  • Spinal cord is again isolated and DNA isolated using three different strategies: (1) whole tissue DNA as in la, (2) flow-sorted nuclei, (3) flow-sorted H2B-mPlum nuclei (contains transduction-competent capsid DNA).
  • NGS is performed and AAV capsids are identified for further screening for spinal cord transduction.
  • FIGs. 2A-C Two-vector iTransduce system allows detection of transduction competent AAV in vitro and in vivo.
  • b Two-vector iTransduce system allows detection of transduction competent AAV in vitro and in vivo.
  • FIGs. 3A-E Isolation and selection of candidate variants in NHPs.
  • a-b Flow cytometry of nuclei isolated from control (a) and library-injected (b) NHPs. Nuclei were selected using fluorescent dye, with mPlum used to discriminate nuclei from transduced cells, c.
  • Candidate variants were selected from whole tissue isolation, mPlum- nuclei, and mPlum+ (transduced) nuclei. The names and insert peptides of each variant are listed, corresponding to SEQ ID NOs:8, 4, 5, 7, 6, 9, 10, 11, 16, 14, 15, and 12.
  • d The frequency of amino acids at each position for the selected candidates is given. Larger size indicates more frequent incorporation of a given amino acid, for a given position, e. Overall production yield of each capsid variant when individually produced, compared to AAV9 (in black).
  • FIGs. 4A-G Barcoded candidate capsid screen identifies variants with enhanced biodistribution in NHP spinal cord and reduced biodistribution to liver.
  • A Schematic showing the procedure used for this experiment. Each capsid variant was barcoded between the protein-coding region and post-transcriptional sequences. Following intrathecal injection, DNA was isolated from 1) each region of the spinal cord, 2) liver, 3) brain, 4) peripheral nerves, and 5) heart. These were subjected to next-generation sequencing, b. Overall biodistribution of AAV capsid genomes in each section of the spinal cord as measured by qPCR. LI, Tl, Cl are from NHP #1001. L2, T2, C2 are from NHP #1002. C.
  • FIGs. 5A-C Barcoded capsid screen identifies variants with enhanced transgene RNA levels in spinal cord and reduced RNA levels in liver, a.
  • Lumbar 1 NHP #1001;
  • Lumbar 2 NHP #1002.
  • Liver 1 NHP #1001;
  • Liver 2 NHP 1002.
  • FIGs. 7A-B Validation of barcoded AAV genomes to detect differences in biodistribution and transgene expression of validate capsids
  • b Quantitation of reads obtained in the liver and brain. Biological replicates are shown for each group. Only one mouse is shown for liver mRNA reads due to a contamination issue with one of the mouse livers. All other samples show two biological replicates. Error bars depict the standard deviation from the mean.
  • FIG. 8 Variant frequency in non-spinal cord tissues of NHPs injected intrathecally with barcoded AAV capsid candidates. Heatmap showing relative frequency of each AAV variant in various regions of the CNS, along with heart and liver. Variants are clustered by expression pattern. Number (1, 2) refers to each animal. Parietal, parietal lobe. Prefrontal, prefrontal cortex. DRG, dorsal root ganglion.
  • FIG. 9 Variant frequency in peripheral nerve tissues of NHPs injected intrathe- cally with barcoded AAV capsid candidates. Heatmap showing relative frequency of each AAV variant in peripheral nerves. Variants are clustered by expression pattern. Number (1, 2) refers to each NHP (#1001 and #1002). 8th, 8th cranial nerve. Sci, sciatic nerve. Su, sural nerve. Ul, ulnar nerve.
  • SCOs human spinal cord organoids
  • c. Quantitation of max-projection images of all organoids per group (n 4-6/capsid).
  • d e.
  • FIGs. 12A-E Analysis of spinal cord organoids transduced with AAV variants (SCO laboratory 2).
  • a Quantification of mean GFP intensity in whole live organoids at days 1, 7, and 10.
  • Quantifications were performed in 3 organoids per hiPSC line per capsid. P values were calculated using 2way ANOVA with Bonferroni’s multiple comparison correction test.
  • b Quantification of mean GFP intensity in whole live organoids at days 1, 7, and 10.
  • CSF cerebral spinal fluid
  • Intrathecal injection into the cerebral spinal fluid (CSF) around the lumbar spinal cord with AAV generally gives good transduction of motor neurons in the lumbar region, but less transduction of cervical regions and the brain 8, 9 .
  • Positioning the subject in the Trendelenburg position can enhance vector biodistribution of the cervical region somewhat 9, 10 .
  • cisterna magna injection of AAV vectors gives better cervical and brain transduction but less so in the lumbar region 11 .
  • AAV9 has been successfully developed for transgene delivery to the spinal cord to treat the neuromuscular disease, spinal muscular atrophy (SMA), there is room for improvement.
  • AAV vectors can cause liver enzyme elevation, complement activation, thrombotic microangiopathy (TMA), and even sepsis, organ failure, and even death 4, 19 .
  • TMA thrombotic microangiopathy
  • sepsis organ failure, and even death 4, 19 .
  • immunosuppressive/modulatory regimens may mitigate some of these serious adverse events 32 , these pharmacological interventions are not without risk and may not be feasible/effective in all patients.
  • much recent research is focused on developing AAV gene therapies for CNS disorders using a CSF route of administration.
  • AAV directly into the CSF has been shown to reduce exposure to neutralizing antibodies which are present at much lower levels than in blood 20 . While there is less systemic exposure to vector than a systemic route of administration, there is still a large amount of vector that can enter the blood and peripheral organs after CSF injection.
  • a recent study by Meseck et al. performed lumbar IT injection of NHPs and measured vector genomes in CNS and peripheral tissues 4 weeks post injection 12 . Remarkably, liver received over 100 times the number of AAV genomes compared to the brain and approximately 10 times more than the spinal cord after lumbar intrathecal injection 12 . Furthermore, transgene expression was higher in the liver and heart compared to spinal cord 12 .
  • the present disclosure is, to the best of the inventors’ knowledge, the first selection of an AAV peptide display library in NHPs using the intrathecal route.
  • the majority of the top capsids identified in our selection showed greatly reduced biodistribution (up to 1,250-fold less frequent reads) and transduction (up to 30,000-fold less frequent reads) in liver compared to AAV9.
  • This desirable feature may allow for less toxicity observed with liver transduction, although this will need to be confirmed by testing with individual capsids encoding transgenes of interest in toxicology studies in NHPs.
  • the top candidate capsids appear to be more efficient than AAV9 at spinal cord transduction at the RNA level (Fig. 5b).
  • RNA-based reads RNA-based reads
  • capsids identified from the whole-tissue isolated DNA Fig. 5b.
  • Whole tissue isolation is likely to contain AAV genomes that may be outside the cell (still in capsids) or nucleus or not uncoated in the nucleus (not transcriptionally active).
  • capsids selected with desired properties in NHPs were effective in a 3D human organoid preclinical model.
  • the human organoid model could serve important roles in preclinical development of gene therapy. First, they could help narrow down the list of candidates from initial pooled barcoded AAV’s such as we did.
  • the human spinal cord organoid data helped us to narrow our top candidates to TR2 being the lead for further expensive NHP testing. Additionally, the organoids could serve in parallel to study a given therapies therapeutic effect if they contain disease-specific mutations (a feature the NHP model does not provide).
  • the human SCO served to validate the capsids which we had chosen from our NHP selections were potent at transduction of clinically relevant cells. Furthermore, these new capsids should serve as very useful gene delivery tools for basic biology studies in spinal cord organoids.
  • capsids TH1 and TR2 yielded statistically significant higher transduction (GFP levels) than AAV9 and thus these capsids should be useful in preclinical gene therapy studies in mice via the intrathecal route.
  • capsids like AAV9, transduced clinically relevant cell types such as neurons and glia. In most cases, transduction efficiency in mice was similar to AAV9, so these capsids may be used for pre-clinical work in mice.
  • capsids While not a primary objective of the study, we also assessed AAV encoded DNA barcode frequency for the pooled capsids in brain and peripheral nerves. From these data, certain capsids appear to outperform AAV9 in biodistribution to certain brain regions and nerves. This includes capsid DH1 in brain and DH1, DPI, and DR2 in nerves. It may be of future interest to test these individual capsids in NHPs after intrathecal injection to assess transduction of brain and nerves.
  • the present methods identified peptide sequences that enhance biodistribution and transduction of spinal cord of an AAV when inserted into the capsid of the AAV, e.g., into AAV1, AAV2, AAV8, or AAV9, or another AAV known in the art or listed herein.
  • the peptides comprise sequences of at least 7 amino acids.
  • the amino acid sequence comprises at least 4, e.g., 5, 6, or 7 contiguous amino acids of the sequences NL1 (LTTEGRR, SEQ ID NOTO), TH1 (HPARALP, SEQ ID NO: 13), TP1 (PKYPLLG, SEQ ID NO: 14), and TR2 (RTTASLM, SEQ ID NO: 12), as well as other capsids listed herein, e.g., in any of Tables III, IV, V, VIA, VIB, or VIC.
  • Peptides including reversed sequences can also be used.
  • Viral vectors for use in the present methods, kits and compositions include recombinant adeno-associated virus (AAV) comprising a capsid peptide as described herein and optionally a transgene for expression in a target tissue (e.g., in an expression construct).
  • AAV adeno-associated virus
  • AAV are a preferred viral vector system for delivery of nucleic acids.
  • AAV is a tiny non-enveloped virus having a 25 nm capsid. No disease is known or has been shown to be associated with the wild type virus.
  • AAV has a single-stranded DNA (ssDNA) genome.
  • ssDNA single-stranded DNA
  • AAV has been shown to exhibit long-term episomal transgene expression, and AAV has demonstrated excellent transgene expression in the brain, particularly in neurons. Space for exogenous DNA is limited to about 4.7 kb.
  • An AAV vector such as that described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells.
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al., Proc. Natl. Acad. Sci. USA 81 :6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51 :611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993).
  • AAV variants over 100 have been cloned
  • AAV variants have been identified based on desirable characteristics.
  • the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AV6.2, AAV7, AAV8, rh.8, AAV9, rh.10, rh.39, rh.43 or CSp3; for CNS use, in some embodiments the AAV is AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, or AAV9. As one example, AAV9 has been shown to somewhat efficiently cross the blood-brain barrier.
  • the AAV capsid can be genetically engineered to enhance biodistribution and transduction of spinal cord, by insertion of a peptide sequence as described herein into the capsid protein, e.g., into the AAV9 capsid protein VP1 between amino acids 588 and 589.
  • An exemplary wild type AAV9 capsid protein VP1 (Q6JC40-1) sequence is as follows:
  • AAV that include one or more of the peptide sequences described herein, e.g., an AAV comprising a capsid protein comprising a sequence described herein, e.g., an AAV9 VP1 capsid protein wherein a peptide sequence described herein has been inserted into the sequence, e.g., between amino acids 588 and 589 (in bold above).
  • the AAV can be, e.g., recombinant episomal AAV.
  • the AAV sequences can be, e.g., at least 80, 85, 90, 95, 97, or 99% identical to a reference AAV sequence set forth herein, e.g., can include variants, preferable that do not reduce the ability of the AAV to mediate transgene expression in a cell.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences can determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web at gcg.com), using the default parameters, e.g., a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the AAV also includes a transgene sequence (i.e., a heterologous sequence), e.g., a transgene encoding a therapeutic agent, e.g., as described herein or as known in the art, or a reporter protein, e.g., a fluorescent protein, an enzyme that catalyzes a reaction yielding a detectable product, or a cell surface antigen.
  • a transgene sequence i.e., a heterologous sequence
  • a transgene encoding a therapeutic agent, e.g., as described herein or as known in the art
  • a reporter protein e.g., a fluorescent protein, an enzyme that catalyzes a reaction yielding a detectable product, or a cell surface antigen.
  • the transgene is preferably linked to sequences that promote/drive expression of the transgene in the target tissue, e.g., in an expression cassette (also referred to herein as an expression construct).
  • transgenes for use as therapeutics include neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial-derived growth factor (GDNF), brain-derived growth factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine hydroxlase (TH), GTP-cyclohydrolase (GTPCH), amino acid decarboxylase (AADC), aspartoacylase (ASP A), blood factors, such as P-globin, hemoglobin, tissue plasminogen activator, and coagulation factors; colony stimulating factors (CSF); interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, etc.; growth factors, such as keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGFs),
  • protein of interest examples include ciliary neurotrophic factor (CNTF); neurotrophins 3 and 4/5 (NT-3 and 4/5); glial cell derived neurotrophic factor (GDNF); aromatic amino acid decarboxylase (AADC); hemophilia related clotting proteins, such as Factor VIII, Factor IX, Factor X; dystrophin or nini -dystrophin; lysosomal acid lipase; phenylalanine hydroxylase (PAH); glycogen storage disease-related enzymes, such as glucose-6-phosphatase, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase (e.g., PHKA2), glucose transporter (e.g., GLUT2), aldolase A, P-enolase, and glycogen synthase; lysosomal enzymes (e.g.,
  • the transgene can also encode an antibody, e.g., an immune checkpoint inhibitory antibody, e.g., to PD-L1, PD-1, CTLA-4 (Cytotoxic T-Lymphocyte- Associated Protein-4; CD 152); LAG-3 (Lymphocyte Activation Gene 3; CD223); TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3; HAVCR2); TIGIT (T- cell Immunoreceptor with Ig and ITIM domains); B7-H3 (CD276); VSIR (V-set immunoregulatory receptor, aka VISTA, B7H5, C10orf54); BTLA 30 (B- and T- Lymphocyte Attenuator, CD272); GARP (Glycoprotein A Repetitions; Predominant; PVRIG (PVR related immunoglobulin domain containing); or VTCN1 (Vset domain containing T cell activation inhibitor 1, aka B7-H4).
  • transgenes can include small or inhibitory nucleic acids that alter/reduce expression of a target gene, e.g., siRNA, shRNA, miRNA, antisense oligos, or long non-coding RNAs that alter gene expression (see, e.g., WO2012087983 and US20140142160).
  • small or inhibitory nucleic acids that alter/reduce expression of a target gene, e.g., siRNA, shRNA, miRNA, antisense oligos, or long non-coding RNAs that alter gene expression (see, e.g., WO2012087983 and US20140142160).
  • transgenes can include genome editing reagents including CRISPR proteins such as CRISPR-Cas9, -Cast 2a nucleases and nickases, cytosine base editors (CBEs), adenine base editors (ABEs), CRISPR prime editors (PEs), variants thereof, and optionally their associated guide RNAs.
  • CRISPR proteins such as CRISPR-Cas9, -Cast 2a nucleases and nickases
  • CBEs cytosine base editors
  • ABEs adenine base editors
  • PEs CRISPR prime editors
  • the expression cassette can also include one or more sequences that promote expression of a transgene, e.g., one or more promoter sequences; enhancer sequences, e.g., 5’ untranslated region (UTR) or a 3’ UTR; a polyadenylation site; and/or insulator sequences.
  • the promoter is a brain tissue specific promoter, e.g., a neuron-specific or glia-specific promoter.
  • the promoter is a promoter of a gene selected from: neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), MeCP2, adenomatous polyposis coli (APC), ionized calcium-binding adapter molecule 1 (Iba-1), synapsin I (SYN), calcium/calmodulin- dependent protein kinase II, tubulin alpha I, neuron-specific enolase and platelet- derived growth factor beta chain.
  • a promoter of a gene selected from: neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), MeCP2, adenomatous polyposis coli (APC), ionized calcium-binding adapter molecule 1 (Iba-1), synapsin I (SYN), calcium/calmodulin- dependent protein kinase II, tubulin alpha I, neuron-specific enolase and platelet- derived growth factor beta chain.
  • the promoter is a pan-cell type promoter, e.g., cytomegalovirus (CMV), beta glucuronidase, (GUSB), ubiquitin C (UBC), or rous sarcoma virus (RSV) promoter.
  • CMV cytomegalovirus
  • GUSB beta glucuronidase
  • UBC ubiquitin C
  • RSV rous sarcoma virus
  • WPRE woodchuck hepatitis virus posttranscriptional response element
  • microRNA miRNA-dependent post-transcriptional suppression of transgene expression can be used to increase specificity of vector-mediated transgene expression.
  • MicroRNAs typically regulate gene expression by binding to sequences in the 3’ untranslated region (UTR) of the mRNA.
  • tandem repeats of artificial microRNA target sites can be incorporated into the 3’ UTR of the transgene expression cassette, leading to subsequent degradation of transgene mRNA in cells expressing the corresponding microRNA, thereby decreasing expression.
  • the AAV also has one or more additional mutations that increase delivery to the target tissue, e.g., the CNS, or that reduce off-tissue targeting, e.g., mutations that decrease liver delivery when CNS, heart, or muscle delivery is intended (e.g., as described in Puierela et al.
  • compositions described herein can be used to deliver any composition, e.g., a deoxyribonucleic acid sequence of interest to a tissue, e.g., to the spinal cord, and central nervous system (brain), heart, muscle, peripheral nervous system (e.g., dorsal root ganglion or peripheral nerves).
  • a tissue e.g., to the spinal cord, and central nervous system (brain), heart, muscle, peripheral nervous system (e.g., dorsal root ganglion or peripheral nerves).
  • the methods include delivery to specific brain regions, e.g., cortex, cerebellum, hippocampus, substantia nigra, amygdala.
  • the methods include intrathecal delivery.
  • the methods include delivery to neurons, astrocytes, or glial cells in the spinal cord or Schwann Cells in the peripheral nerves.
  • the methods and compositions are used to deliver a nucleic acid sequence to a subject who has a disease, e.g., a disease of the CNS; see, e.g., US9102949; US 9585971; and US20170166926.
  • the subject has a condition listed in Table A; in some embodiments, the vectors are used to deliver a therapeutic agent (e.g., sequence encoding the target (gene addition therapy) or inhibitory nucleic acid that reduces expression of the target) listed in Table A for treating the corresponding disease listed in Table A.
  • a therapeutic agent e.g., sequence encoding the target (gene addition therapy) or inhibitory nucleic acid that reduces expression of the target
  • the therapeutic agent can be delivered as a nucleic acid, e.g., via a viral vector, wherein the nucleic acid encodes a therapeutic protein or an inhibitory nucleic acid such as an antisense oligo, siRNA, shRNA, or artificial miRNA that reduces expression of the target, and so on; or as a fusion protein/complex with a peptide as described herein.
  • a nucleic acid e.g., via a viral vector, wherein the nucleic acid encodes a therapeutic protein or an inhibitory nucleic acid such as an antisense oligo, siRNA, shRNA, or artificial miRNA that reduces expression of the target, and so on; or as a fusion protein/complex with a peptide as described herein.
  • compositions described herein can be used to treat these conditions in a subject in need thereof, by administration of a therapeutically effective amount of an AAV carrying a therapeutic transgene, sufficient to ameliorate, reduce risk of, or delay onset of one or more symptoms of the condition.
  • compositions comprising the AAVs as an active ingredient.
  • compositions typically include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • compositions are typically formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intraarterial, subcutaneous, intraperitoneal, intrathecal, intramuscular, or injection or infusion administration. Delivery can thus be systemic or localized.
  • parenteral e.g., intravenous, intraarterial, subcutaneous, intraperitoneal, intrathecal, intramuscular, or injection or infusion administration. Delivery can thus be systemic or localized.
  • delivery into the cochlea through application over or through the round window membrane, through a surgically drilled cochleostomy adjacent to the round window, a fenestra in the bony oval window, or a semicircular canal can be used (see, e.g., Kim et al., Mol Ther Methods Clin Dev.
  • subretinal or intravitreal injections can be used (see, e.g., Ochakovski et al., Front Neurosci. 2017; 11 : 174; Xue et al., Eye (Lond). 2017 Sep;31(9):1308-1316).
  • solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.
  • the kit can include compositions comprising an AAV comprising a peptide as described herein.
  • Human 293 T cells were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in high glucose Dulbecco’s modified Eagle’s medium containing HEPES (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Sigma, St. Louis, MO) and 100 U/mL penicillin, 100 pg/mL streptomycin (Invitrogen) in a humidified atmosphere supplemented with 5% CO2 at 37 °C.
  • HEPES high glucose Dulbecco’s modified Eagle’s medium containing HEPES (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Sigma, St. Louis, MO) and 100 U/mL penicillin, 100 pg/mL streptomycin (Invitrogen) in a humidified atmosphere supplemented with 5% CO2 at 37 °C.
  • FBS fetal bovine serum
  • pAAV-CBA-Floxed-STOP-H2B-mPlum plasmid was constructed by digesting the AAV ITR containing plasmid, AAV-CBA-WPRE, after the CBA promoter with Hindlll and Nhel restriction sites.
  • a DNA fragment for a floxed transcription stop transcription site (3 copies of SV40 late poly A sequence) followed by a H2B protein fused to mPlum was synthesized by Genscript (Piscataway, NJ) and inserted into pUC57-Kanamycin plasmid.
  • pUC57-Kan with Floxed-STOP-H2B- mPlum was digested with Hindlll and Nhel and inserted into the above digested AAV plasmid to create pAAV-CBA-Floxed-STOP-H2B-mPlum (plasmid electronic map available upon request).
  • pAAV-CBA-hFrataxin-HA-BC-WPRE plasmid was constructed by digesting the AAV transgene expression plasmid, pAAV-CBA-GFP, with Agel and Nhel to remove the GFP cDNA fragment.
  • a double stranded DNA gBlockTM fragment was ordered from Integrated DNA Technologies (IDT, Coralville, Iowa) which contained human frataxin cDNA fused with a hemagglutinin (HA) tag on its C-terminus.
  • This fragment was inserted into the pAAV plasmid above using the Gibson Assembly® Master Mix (New England Biolabs, Ipswich, MA).
  • This “base” plasmid, pAAV- CBA-hFrataxin-HA was used as the acceptor plasmid to insert barcode fragments downstream of the HA sequence (plasmid map available on request).
  • pAAV-CBA-hFrataxin-HA was digested with Xhol.
  • double stranded DNA gBlockTM fragments 130 bp
  • IDT double stranded DNA gBlockTM fragments
  • capsid candidates for 14 capsid candidates as well as AAV9
  • flanking homology arms were individually cloned into the Xhol-digested pAAV-CBA-hFrataxin-HA plasmid using Gibson Assembly as before.
  • Each barcoded pAAV-CBA-hFrataxin-HA plasmid was complete-plasmid sequenced at PlasmidSaurus (Eugene, OR).
  • AAV 9 capsid candidates containing unique 7mer peptides were individually cloned into the AAV9 rep/cap plasmid, pAR9, as previously described 16 .
  • the plasmid AAV-CBA-GFP has been previously described 38 and was used in the individual capsid comparisons in mice and in human spinal cord organoids.
  • This AAV expression plasmid encodes a single stranded AAV genome with inverted terminal repeat (ITR)-flanked transgene cassette with a CMV IZE enhancer, chicken beta actin promoter, a chimeric intron, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and tandem bovine growth hormone (BGH) and SV40 polyA signal sequences.
  • ITR inverted terminal repeat
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • BGH tandem bovine growth hormone
  • AAV9 iTransduce library was performed as previously described 16 with some changes. Fifty tissue culture dishes (15 cm diameter) were used (1.5xl0 7 293T cells seeded per plate), with cells cultured in DMEM containing 10% fetal bovine serum (FBS) and 100 U/mL of penicillin, 100 pg/mLstreptomycin, and 292 pg/mL L-glutamine (Invitrogen).
  • FBS fetal bovine serum
  • penicillin 100 pg/mLstreptomycin
  • 292 pg/mL L-glutamine Invitrogen
  • the pooled virus was then purified by iodixanol density-gradient ultracentrifugation. Buffer exchange to phosphate buffered saline (PBS) containing 0.001% Pluronic F-68 (Gibco) was done using ZEBA spin columns (7K MWCO; Thermo Fisher Scientific) and further concentration was performed using Amicon Ultra lOOkDa MWCO ultrafiltration centrifugal devices (Millipore).
  • PBS phosphate buffered saline
  • Pluronic F-68 Pluronic F-68
  • Endotoxin for both vector preparations was determined to be ⁇ 1 EU/mL using Endosafe® LAL Cartridges and the Endosafe® nexgen PTSTM device (Charles River, Washington, SC). Vector purity was assessed by silver staining of SDS PAGE gels in which IxlO 10 vg of each vector was run/lane. Purity of both preparations were >90%. Vector was stored at -80°C until use.
  • AAV-CBA-Floxed-STOP-H2B-mPlum production This vector was produced, purified, and titered by Vector Biolabs (Malvern, PA/ Endotoxin level was below 1 EU/mL.
  • pAAV-CBA-hFrataxin-HA- barcode (6 pg)
  • pAR9-peptide of interest 7 pg
  • pAdAF6 helper plasmid, 15 pg.
  • AAV was isolated from a pool of clarified cell lysate and polyethylene glycol (PEG)-precipitated vector from the conditioned media. The pooled virus was then purified by iodixanol density-gradient ultracentrifugation.
  • AAV production was identical to that described in “barcoded AAV capsid candidate production” above with the exception that the expression plasmid was AAV-CBA-GFP.
  • the animal received an IT dose of unselected AAV library as appropriate to group (per the study design Table I).
  • the dose volume injected was 0.74 mL and vector dose of 9xlO n vg.
  • the animal was given Buprenorphine (0.03 mg/kg, IM) and Meloxicam (0.2 mg/kg, SC) prior to the procedure for the purpose of analgesia.
  • the animal was sedated with Ketamine 7.5-12 mg/kg and Dexdomitor 0.01-0.03 mg/kg mixture, IM; Atipamezole (0.1-0.3 mg/kg, IM) was used for reversal.
  • the animal was positioned in lateral recumbency while on a circulating warm water blanket and/or forced warm air blanket during the procedure.
  • the head was kept in line with the spine and the hips and shoulders were perpendicular to the table. The lower back was arched to increase spacing between the spinous processes.
  • the lumbosacral region (the area over -L4/5 for cynomolgus) was clipped and aseptically prepared utilizing 3 alternating scrubs of either povidone iodine or chlorhexidine scrub solution and sponges soaked in 70% isopropyl alcohol.
  • a line block i.e., Lidocaine/Bupivacaine
  • SC was administered at the lumbar puncture site.
  • a final application of ChloraPrepTM or appropriate antimicrobial was applied to the puncture site and allowed to dry.
  • the wings of the ileum were palpated to provide anatomical landmarks.
  • the two spinous processes were identified, and in between which the spinal needle (22 g x 1.5") was introduced.
  • the skin was penetrated and needle slowly advanced. After confirmation of placement in the intrathecal space, -0.5 mL of CSF was removed prior to dose administration.
  • the AAV vector was slowly administered over 1-2 minutes. After administering the dose, the syringe and needle was left in place for -5 seconds and after removal, pressure applied to the injection site. Parameters were observed constantly throughout the procedure including heart rate, respiratory rate and oxygen saturation. Animals were recovered from anesthesia and moved to a recovery area, placed on a circulating warm water blanket and/or forced warm air blanket and covered with a dry towel.
  • mice were observed following the procedure and kept in the recovery area until the animal was conscious and able to hold itself in a sitting position. Animals were then transported to their home cage. Approximately 2 hours post vector dosing, animals were immunosuppressed with intramuscular dosing of 0.5 mg/kg of dexamethasone, which continued daily until necropsy at day 21.
  • Round 2 selection The IT injection and animal care was identical to round 1 with the following changes.
  • mice were anesthetized with Ketamine 7.5-12 mg/kg and Dexdomitor 0.01-0.03 mg/kg mixture. Nembutal was administered at 15-30 mg/kg. Once deeply anesthetized, the animal was perfused via left cardiac ventricle with cold heparinized (100 U/mL) saline until the outflow ran clear. Approximately 1 L of heparinized saline was used with a perfusion pump set to -400 rpm. Euthanasia was performed per AMVA guidelines. For rounds 1 and 2, flash frozen samples were collected for the entire spinal cord and brain and stored at -80°C.
  • the homogenate containing nuclei was layered on top of the iodixanol gradient and centrifuged at 10,000xg at 4°C for 30 min in a fixed angle rotor (FA-45-6-30, Eppendorf, Enfield, CT). The gradient was carefully aspirated leaving the nuclei pellet in the tube. Nuclei were resuspended in 500 pl of cold PBS. Next nuclei were labeled with VybrantTM Dy eCycleTM Violet Stain (ThermoFisher) and were sorted on a BD FACS Aria II Cell Sorter (Becton Dickinson, Franklin Lake, NJ). First, violet stain positive nuclei were gated on (PI) using Pacific Blue-A laser.
  • Next generation sequencing was performed on the plasmid AAV9 library pool, as well as following packaging of capsids.
  • packaged naive library was amplified by PCR and sequenced at a depth of -105 reads to ensure adequate read depth and a lack of bias. Minimum requirements were >95% unique variant reads, and no single variant appearing in more than 10 sequencing. Sequencing was also performed following PCR rescue of the cap gene fragment (either from NHP spinal cord tissue or from nuclei sorted by flow cytometry).
  • Sequence output files were quality-checked initially using FastQC (bioinformatics.babraham.ac.uk/projects/fastqc/) and analyzed on a program custom- written in Python. Briefly, sequences were binned based on the presence or absence of insert; insert-containing sequences were then compared to a baseline reference sequence and error-free reads were tabulated based on incidences of each detected unique insert. Inserts were translated and normalized.
  • Non-human primate candidate barcoded library screen Two adult male cynomolgus monkeys (see Table I for NHP information) were intrathecally injected in the same manner as for the selection, except with 4.48xl0 12 vg of the pooled barcoded capsids. Three weeks later, animals were perfused with sterile heparinized saline. Samples for paraffin embedding were fixed in 10% neutral buffered formalin before transferring the samples to PBS. Samples for cryosectioning were fixed in 4% buffered formaldehyde for 24 h before transferring samples to PBS. Samples for DNA and RNA extraction were immediately frozen on dry ice and stored at -80°C.
  • DNA isolation Whole tissue isolation. Spinal cord (and other tissue) samples (0.5 cm-1 cm in length) were homogenized using 1.4 mm ceramic beads in a BeadBug tissue homogenizer (Benchmark Scientific) in buffer ATL of the DNeasy Blood and Tissue Kit (Qiagen). After homogenization, we followed the manufacturer’s instructions to purify DNA. DNA concentration was determined using a NanoDrop spectrophotometer (ThermoFisher Scientific).
  • RNA isolation Spinal cord (and other tissue) was placed into Qiazol (Qiagen) and subjected to homogenization with 1.4 mm ceramic beads in a BeadBug tissue homogenizer (Benchmark Scientific). After performing a phenol/chloroform extraction of RNA, the RNA was precipitated using isopropranol and centrifugation, followed by a 75% ethanol wash. The RNA pellet was resuspended in RNase-free water and stored at -80C until further processing. The RNA sample was further purified using RNeasy spin columns (Qiagen) and the sample was eluted in RNase- free water.
  • RNA was synthesized from RNA (approximate input 500 ng of RNA) using the SuperScriptTM IV VILOTM Master Mix reverse transcriptase (RT) kit.
  • RT SuperScriptTM IV VILOTM Master Mix reverse transcriptase
  • Quantitative PCR to measure absolute amounts of AAV genomes and transgene mRNA in spinal cord of pooled AAV injection in NHPs. DNA and RNA isolation and cDNA synthesis from NHP spinal cord was performed as described immediately above. To determine the amounts of AAV vector genomes or transgene expression (frataxin-HA) we used the same Taqman probes and primers targeting BGH polyA used to titer our AAV vectors. For AAV vector genomes, we used a standard curve with an AAV plasmid to perform absolute quantitation. We used 54 ng of genomic DNA template from lumbar, thoracic, and cervical spinal cord as input for the qPCR.
  • a separate qPCR reaction for each sample was performed to detect the NHP gene UBE2D2 (ubiquitin conjugating enzyme E2 D2) using the Taqman Gene Expression Assays 20x mix (Catalog #4351372, assay ID Mfi)7285893_sl, ThermoFisher Scientific).
  • AAV genomes were inferred from the AAV plasmid standard curve and adjusted to AAV genomes/pg of genomic DNA.
  • For each AAV genome sample we calculated the 2 Adelta Ct (sample Ct - sample with lowest Ct) of the UBE2D2 qPCR sample and then normalized the AAV genomes to that value. This compensated for DNA input differences for each sample.
  • RT-qPCR samples we used the AAV plasmid as a standard curve as above to calculate absolute amounts of cDNA.
  • a separate qPCR reaction for each sample was performed to detect the NHP cDNA of GAPDH (glyceraldehyde-3 -phosphate dehydrogenase) using the Taqman Gene Expression Assays 20x mix (assay ID Mf04392546 gl, ThermoFisher Scientific). This probe spans exons, so it is selective for cDNA detection over genomic DNA.
  • cDNA values for AAV transgene were normalized to the respective GAPDH Ct values for each sample to control for differences in input total cDNA. All qPCR reactions were performed in a 7500 FAST qPCR system (Applied Biosystems).
  • Quantitative PCR to measure absolute amounts of AAV genomes in spinal cord organoids.
  • DNA was extracted and purified from organoids using the DNeasy Blood and Tissue Kit (Qiagen).
  • the same Taqman probe and primer set described above was used for AAV genomes.
  • human cDNA detection used for normalization we used a probe and primer set specific for the human ubiquitin conjugating enzyme E2 D3 (UBE2D3) Taqman Gene Expression Assays 20x mix (Assay Id Hs00704312_sl, ThermoFisher Scientific).
  • PCR to amplify barcodes for NGS.
  • DNA and RNA samples from the pooled barcoded library injected animals we used the following reagents.
  • a PCR using either the DNeasy -purified DNA or the cDNA from the RT reaction as template was performed (usually 150 ng DNA template input).
  • NEB Q5 polymerase
  • primers that amplify a 182bp amplicon which contains the unique barcode for each capsid.
  • Primers were: SC-Lib-Fwd (5’ gatgcttacccttacgacgt 3’) and SC-Lib-Rev (5’ cagcgtatccacatagcgta 3’).
  • the PCR product was purified using the PureLinkTM PCR Purification Kit (ThermoFisher Scientific) and samples were submitted for NGS as described above. We initially sequenced an aliquot of the pooled barcoded library to ascertain the precise frequencies of the barcoded variants on input. Output frequencies in all tissues were normalized to these initial ratios. Insert-containing sequence reads were binned using Hamming distance, comparing to the known barcode sequences; additional quality control was undertaken to ensure only true barcoded reads were assigned to each capsid.
  • the tip of the needle was introduced into the lumbar spine, and proper insertion of the needle was confirmed by tail flick reflex. After slowly injecting the total dose volume, the needle will be allowed to stay in place for a minimum of 5 seconds to avoid efflux of the test material to periphery. Animals were placed on a circulating water heating pad until fully recovered from anesthesia. Four weeks after administration, animals were euthanized (carbon dioxide asphyxiation, to effect, in conjunction with exsanguination), and tissues were removed for fixation (histology) or snap frozen in liquid nitrogen.
  • Nonspecific protein-protein interactions were blocked with Leica PowerVision IHC/ISH Super Blocking solution (Leica PV6122) at RT.
  • the primary rabbit polyclonal anti-GFP antibody Cat. no. Al 1122, ThermoFisher
  • CST Cell Signaling Technology
  • a biotin-free polymeric IHC detection system (Leica DS9800) consisting of HRP conjugated Gt anti-Rb IgG secondary antibody was applied for 25 minutes at RT. Immunoreactivity was detected with the diaminobenzidine (DAB) chromogen reaction.
  • DAB diaminobenzidine
  • GFP enzyme-linked immunosorbent assay of spinal cord from mice injected intrathecally with AAV capsids packaging AAV-CAG-GFP cassette.
  • a GFP ELISA kit (abeam ab 171581) was used to detect AAV vector encoded GFP in spinal cord homogenates. Flash-frozen spinal cord segments (each spinal cord was divided into a caudal and rostral segment/mouse) were cut into pieces weighing approximately 5-20 mg and placed into individual tubes with a single stainless-steel bead. Next, we added cell extraction buffer from the kit to each tube and homogenized the tissue in a BeadBugTM benchtop tissue homogenizer (Benchmark Scientific, Atkison, NH) for 5s.
  • BeadBugTM benchtop tissue homogenizer Benchmark Scientific, Atkison, NH
  • hESCs H9 were grown in the mTeSRl (STEMCELL Technologies) medium in 35 mm-diameter tissue culture dishes coated with Matrigel (Coming, 354277; 1 :25 in DMEM/F12). hESCs were detached from 35 mm-diameter dishes using ReLeSR (STEMCELL Technologies), and cell aggregates in mTeSR were added to each well of a 12-well micropattern plate.
  • mTeSR medium was replaced with the neural cell induction medium (DM, DMEM/F12, 1% N2 supplement, 2% B27 supplement, 1% NEAA, 1% penicillin, and 0.1% 2-mercaptoethanol) containing 10 pM SB431542 (R&D) and 3 pM CHIR99021 (Sigma), and was subsequently replaced every day.
  • colonies were detached using the pressure of a cell recovery solution (Corning, Cat#.354253) from the pipette under a stereoscopic microscope and transferred into an Ultra-Low Attachment 96-well clear round bottom plate (Corning- Costar). Further, they were further grown in 3D with DM containing 20 ng/mL bFGF.
  • organoids were transferred to 6 well plate and grown while shaking 80 rpm.
  • organoids of 66 days or 225 days were transferred into an Ultra-Low Attachment 96-well plate.
  • 35 pl of 1.17X10 11 gc of AAV in PBS containing 10% sucrose was added to each organoid 33 .
  • the organoids were transferred to 6 well plate and incubated while shaking 80 rpm.
  • live images of each organoid were taken in 5- pm steps along the z-axis using confocal microscope (Leica TCS SP8 confocal microscope). Stacked images using Z stack maximal projection were used for quantification using ImageJ.
  • samples were incubated with primary antibodies in a blocking buffer overnight at 4°C.
  • Samples were washed three times with PBST (0.2% Triton X-100 in PBS) and incubated with secondary antibodies, with Hoechst for cell nuclei staining (1 :2,000), in the blocking buffer for 1 h at 25 °C.
  • PBST PBST
  • samples were mounted in Crystal Mount (Biomed) and imaged using a confocal microscope (Leica TCS SP8 confocal microscope).
  • the original TIFF files were loaded into ImageJ and the channels were split into two windows.
  • the Slice Keeper Image>Stacks>Tools>Slice Keeper
  • the brightness and contrast for the DAPI stack was adjusted to be able to visualize the whole perimeter of the organoid for each slice while all the slices in the GFP stack were set to the same minimum and maximum displayed value (Min: 12 and Max:54).
  • the windows needed to be synchronized (Analyze>Tools>Synchronize Windows). After synchronizing the windows, the freehand selection cursor was used to outline the signal of the DAPI channel. The following measurements were acquired: Area, Mean Gray Value, Min & Max Gray Value and Integrated Density. All data was saved in Excel spreadsheets.
  • hiPSC colonies cultured on vitronectin (Stem Cell Technologies, #7180) and in TeSRTM-E8TM medium (Stem Cell Technologies, #5940) were dissociated with Accutase (MERCK, #SF006) at 37°C for 7 min., made into a single-cell suspension, and 9,000 cells were plated into individual ultra-low attachment U-bottom 96-well plates (Corning, #CLS7007) in 150pl of TeSRTM-E8TM medium (Stem Cell Technologies, #5940) supplemented with ROCK inhibitor (lOpM; Selleckchem, #S1049) and FGF2 (4ng/ml; Peprotech, #AF-100- 18B).
  • TeSRTM-E8TM medium supplemented with the two SMAD inhibitors dorsomorphin (2.5pM; Selleckchem, #S7306) and SB431542 (lOpM; Selleckchem, #S1067) was added. Two thirds of this medium was refreshed daily for the next five days, supplementing with CHIR 99021 (3pM; Cayman Chemicals, #13122) from day 6 to 20.
  • NMM Neural Maintenance Medium
  • 1 DMEM/F12 Thermo Fisher Scientific, #11330
  • Neurobasal Medium Thermo Fisher Scientific, #10888
  • 0.5% N2 Thermo Fisher Scientific, #175020
  • 1% B27 Thermo Fisher Scientific, #17504
  • human insulin 5pg/ml
  • MERCK 1% B27
  • human insulin 5pg/ml
  • L-glutamine 1.5mM
  • Thermo Fisher Scientific, #25030 non-essential amino acids
  • lOpM Thermo Fisher Scientific, #11140
  • B-mercaptoethanol lacicillin-streptomycin
  • penicillin-streptomycin lacicillin-streptomycin
  • hiPSC-derived motor neurons hiPSC-motor neurons were generated following 40 with the following modifications. Briefly, hiPSC colonies cultured on vitronectin (Stem Cell Technologies, #7180) and in TeSRTM-E8TM medium (Stem Cell Technologies, #5940) were dissociated with Accutase (MERCK, #SF006) at 37°C for 5 min., made into a single-cell suspension, and 9,000 cells were plated into individual ultra-low attachment U-bottom 96-well plates (Corning, #CLS7007) in 150pl of TeSRTM-E8TM medium (Stem Cell Technologies, #5940) supplemented with ROCK inhibitor (lOpM; Selleckchem, #S1049) and FGF2 (4ng/ml; Peprotech, #AF-100-18B).
  • MNP1 was changed to MNP2 consisting of NMM supplemented with CHIR (IpM), 2pM SB-431542 (2pM), Dorsomorphin (0.2pM), RA (0.1 pM, ReproCell, #04-0021), SAG (0.25pM; Cayman Chemicals, #11914), and Ascorbic acid (O.lmM).
  • CHIR IpM
  • 2pM SB-431542 2pM
  • Dorsomorphin 0.2pM
  • RA 0.1 pM, ReproCell, #04-0021
  • SAG 0.25pM
  • Cayman Chemicals, #11914 Cayman Chemicals, #11914
  • Ascorbic acid O.lmM
  • MNP1 media supplied with lOpM ROCK inhibitor on the plating day.
  • patterned progenitors were dissociated into single cells with Accutase at 37°C for 5 min. and plated on Poly-L-Omithine and laminin coated glass coverslips at a cell density of 35,000 cells /cm 2 in motor neuron differentiation (MN) media consisting of NMM supplemented with RA (0.5pM), SAG (0.1 pM), Ascorbic acid (O.
  • MN motor neuron differentiation
  • organoids were washed with PBS, fixed in 4% paraformaldehyde (PFA in dEEO, ProSciTech, #C004) at room temperature for 30 min., and followed by sucrose cry opreservation (30% sucrose in PBS at 4°C for 48h), embedding in OCT (Tissue-Tec Oct Compound, #4583) and snap-freezing.
  • PFA paraformaldehyde
  • OCT tissue-Tec Oct Compound, #4583
  • snap-freezing For IHC, 20pm thick sections were cut using a cryostat (Leica). After 6x PBS washes of 5 min., antigen retrieval was performed by incubating slides in 0.01M citrate buffer at 90°C for 30 min.
  • cells were washed 6x 5 minutes with PBS, incubated with diamidino-2-phenylindole (1 : 1000; DAPI; MERCK, #D9542) for 2 minutes at room temperature, washed once with PBS and embedded with Fluoromount-G (Southern Biotech, #0100-01).
  • the AAV genomes consists of a promoter driving a Cre-recombinase cassette as well as p41 promoter driven AAV9 capsid with 7-mer peptide inserts between amino acids 588-589 of VP1. This allows surface display of 50 copies of peptides on VP3 on the capsid surface (and 5 copies each on internally localized VP1 and VP2 proteins).
  • AAV peptide display library For round one ofour in vivo selection strategy in NHPs, we produced the AAV peptide display library and injected it intrathecally into Old World monkeys (cynomolgus macaques) (Fig. la, Table I).
  • AAV genome DNA was recovered by PCR from cervical, thoracic, and lumbar regions of the spinal cord and subjected to next generation sequencing (NGS) to analyze the content of the 7-mer encoding inserts.
  • NGS next generation sequencing
  • These capsid inserts were pooled and packaged into the round 1 selected spinal cord library (Fig. la).
  • Fig. la For the second selection round, we developed a strategy to enable selection of capsids that could mediate transduction expression in a non-transgenic animal such as an NHP. To do this we used a two-vector approach as outlined in Fig. lb.
  • the first vector is the AAV iTransduce library genome shown in Fig. la, that is packaged inside the round 1 rescued capsid inserts.
  • the second vector is a Floxed reporter cassette encoding an H2B-fused mPlum protein called AAV- CBA-Floxed-STOP-H2B-mPlum.
  • H2B-mPlum mPlum with a nuclear localization signal
  • a preparation of the iTransduce AAV9 peptide display library was produced, purified, and titered.
  • Library diversity was determined by NGS to be adequate for the selection and a male cynomolgus monkey was injected intrathecally with 9xlO n vector genomes (vg) of the AAV library.
  • vg 9xlO n vector genomes
  • the animal was killed, perfused with sterile saline and tissues including the spinal cord were flash frozen and stored at -80°C.
  • Example 3 Two vector system allows detection of transduction competent AAV capsids in non-transgenic large animals.
  • AAV-CBA-Floxed-STOP-H2B-mPlum as described in Fig. lb.
  • AAV-PHP.B-CBA-Cre only
  • AAV9-floxed-STOP-H2B-mPlum only
  • mixture of AAV-PHP.B-CBA-Cre with AAV9-floxed-STOP-H2B-mPlum only
  • mice were perfused with PBS and brains flash frozen.
  • nuclei from dissociated brain, labeled total nuclei with a violet-fluorescing dye, and then analyzed nuclei from each group from mPlum fluorescence (Fig 2b). All groups had nuclei, as observed by the violet dye stain.
  • AAV- Floxed-STOP-H2B-mPlum+ AAV-Cre group showed many fluorescent mPlum+ nuclei, which demonstrates the specificity and functionality of the two-vector system (Fig. 2c). The flow data was confirmed in brain sections from the different injected groups.
  • Example 4 Round 2 of selection enriches for capsids that are maintained in the NHP spinal cord after intrathecal injection
  • lb we isolated AAV genomes containing the 21 bp inserts via three methods: (1) whole tissue as in round 1, (2) sorting dye-labeled nuclei from dissociated spinal cord, and (3) sorting H2B-mPlum positive nuclei from cells cotransduced by a transduction competent AAV capsid (Cre-expressing) and the AAV9- Floxed-STOP-H2B-mPlum vector.
  • whole tissue isolated DNA we used samples from all three regions of the spinal cord as in round 1.
  • For the nuclei isolation we used the lumbar region of spinal cord to increase the chances of detecting mPlum expression as this was the injection region of vector and should have the highest vector concentration.
  • the top peptides were enriched by 25 to 67-fold (Tables VIA-C). For the mPlum positive and negative nuclei there were far fewer variants.
  • Example 5 Barcoded capsid screen reveals variants with enhanced biodistribution in spinal cord and lowered liver biodistribution compared to AAV9.
  • capsids had higher levels of genomes in thoracic (up to 30-fold) and cervical (up to 10-fold) regions compared to AAV9, although there was more inter-animal variability compared to the lumbar region (Fig. 4d and 4f). Notably, biodistribution to the liver was reduced for the majority of tested capsids (Fig. 4c), which ranged from 2- to 1,250-fold lower than AAV9 (Fig 4e). NGS barcode analysis of DNA from heart also suggested a lower biodistribution of most of the capsids to peripheral organs compared to AAV9 (Fig 4g).
  • capsids DPI and DR2 had high read frequencies in sural and ulnar nerves compared to AAV9, DH1 had higher levels in sciatic nerve and 8 th cranial nerve, and DK1 in 8 th cranial nerve.
  • Example 6 Enhanced transduction in spinal cord and lower transduction of liver with several candidate capsids
  • cDNA reverse transcribed from the frataxin-HA cDNA was assessed.
  • the cDNA levels were detected in both animals in the lumbar region, although the levels were significantly higher (12.5-fold) in NHP #1001 than #1002 (Fig. 5a). This could be due to differences in transduction efficiency between animals, as the vector genomes were quite similar between animals (Fig 4b).
  • the levels of cDNA in thoracic and cervical regions were not above the negative controls, likely owing to the low injected dose of each capsid, so further analysis of mRNA/cDNA was focused on the lumbar region of spinal cord and the liver.
  • NGS was performed on the barcode cDNA from mRNA isolated from lumbar spinal cord and liver.
  • At the RNA level far fewer capsids outperformed AAV9. None of capsids from the whole tissue DNA isolation outperformed AAV9 in at least one of the injected animals by a factor of >2.
  • 1 of 2 in the nuclei isolation did, and 3 of 5 in the mPlum+ nuclei isolation did (Fig. 5b).
  • the average enhancement ranged from 1.8 to 2.4-fold for the four capsids over AAV9 and up to 3.5-fold in one of the two animals (Fig. 5b). Liver RNA expression appeared similar to the DNA biodistribution data; capsid variants showed expression levels 2 to 30,000-fold lower compared to AAV9 (Fig. 5c).
  • Example 7 Generating a profile of candidate capsid features allows identification of top candidates for preclinical development
  • NHP- selected capsids were also functional in mice.
  • AAV capsids selected in NHP were “backwards compatible”
  • adult 6-8 week old C57BL/6 mice were injected intrathecally in the lumbar region individually with either AAV9, NL1, TH1, TP1, or TR2 (Fig. 6a).
  • Caudal and rostral spinal cord segments were homogenized and a GFP ELISA was conducted.
  • Example 9 Enhanced transduction by NHP-selected capsids in human spinal cord organoids
  • SCOs human spinal cord organoids
  • TR2 had the highest number of GFP+ cells in the top and middle slices, being 2.9-fold and 2.1-fold higher than AAV9, respectively (Fig. llh).
  • TH1 had the highest GFP+ count at the bottom slice, being 2.1 -fold higher than AAV9 (Fig. llh).
  • AAVrhlO vector corrects pathology in animal models of GM1 gangliosidosis and achieves widespread distribution in the CNS of nonhuman primates. Molecular Therapy- Methods and Clinical Development. DOI: doi. org/10.1016/j.omtm.2022.10.004
  • AAV9 intracerebroventricular gene therapy improves lifespan, locomotor function and pathology in a mouse model of Niemann-Pick type Cl disease. Hum. Mol. Genet. doi: 10.1093/hmg/ddy212.

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Abstract

L'invention concerne des capsides d'AAV modifiées ayant une biodistribution et une efficacité de transduction améliorées dans la moelle épinière et une biodistribution réduite dans le foie, des compositions comprenant les capsides, et des procédés d'utilisation de celles-ci.
PCT/US2024/024996 2023-04-17 2024-04-17 Capsides d'aav pour administration sur cible à la moelle épinière Ceased WO2024220545A2 (fr)

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