Described herein are engineered AAV vectors for transgene expression, e.g., in the CNS, PNS, inner ear, heart, or retina, and methods of use thereof. Also provided are methods for discovering new engineered AAV vectors that mediate transgene expression in desired cell types.
While AAV9 vectors have shown remarkable potential for delivery to the CNS after systemic delivery, resulting in clinical success in pediatric patients with spinal muscular atrophy1, systemic injection of high doses of AAV vectors can lead to induction of a T-cell response that can eliminate transduced cells2. In monkeys, there is one report in which high systemic doses of an AAV9-like vector resulted in toxicity and death of the animal which was attributed to systemic inflammation3. A recent phase I clinical trial using high dose AAV9 to treat muscular dystrophy was placed on hold by the FDA due to an immune reaction after vector infusion in one patient. The reason high doses are required is due to the relatively low efficiency of AAV on a per-vector genome copy basis to provide adequate transgene expression in a substantial number of target cells. Thus, developing new AAV capsids which allow more efficient transduction at lower doses should result in better therapeutic efficacy while lowering safety issues, such as immunotoxicity.
Described herein are methods that use an adeno-associated virus (AAV) vector genome with a two-part expression cassette to identify novel virus clones. The first is a Cre-recombinase cassette under a promoter of interest. The second part is an AAV promoter to drive expression of an engineered capsid gene, cloned “in cis” to the first section of the viral genome. Virus vectors are selected for transgene expression (highly sensitive Cre expression) using cells that express a reporter gene (e.g., green fluorescent protein) with an upstream loxP/stop site, thus preventing reporter expression until AAV vector-delivered Cre removes the stop site. Reporter gene positive cells can be isolated and recovered AAV capsid sequences will have a higher likelihood of mediating efficient transgene expression. Also described herein are engineered viral sequences that drive efficient expression in the central nervous system (CNS) and peripheral nervous system (PNS), heart, liver, and inner ear.
Thus, provided herein are AAV capsid proteins comprising an amino acid sequence that comprises at least four contiguous amino acids from the sequence STTLYSP (SEQ ID NO: 1) or FVVGQSY (SEQ ID NO:2). In some embodiments, the AAV capsid proteins comprise an amino acid sequence that comprises at least five contiguous amino acids from the sequence STTLYSP (SEQ ID NO:1) or FVVGQSY (SEQ ID NO:2). In some embodiments, the AAV capsid proteins comprise an amino acid sequence that comprises at least six contiguous amino acids from the sequence STTLYSP (SEQ ID NO: 1) or FVVGQSY (SEQ ID NO:2). Alternatively, the AAV capsid proteins comprise an amino acid sequence that comprises at least four, five, or six contiguous amino acids from the sequences shown in
In some embodiments, the AAV is AAV9.
In some embodiments, the AAV capsid proteins comprises AAV9 VP1.
In some embodiments, the sequence is inserted into the capsid at a position corresponding to amino acids 588 and 589 of SEQ ID NO:6, at the VP1/VP2 interface (amino acid 138) or any site between 583-590.
Also provided herein are nucleic acids encoding an AAV capsid protein as described herein.
Additionally, provided herein are AAVs comprising the capsid proteins described herein, and preferably not comprising a wild type VP1, VP2, or VP3 capsid protein. In some embodiments, the AAVs further comprising a transgene, preferably a therapeutic transgene.
Further provided are methods of delivering a transgene to a cell, e.g., a cell in vivo or ex vivo/in vitro. The methods include contacting the cell with an AAV as described herein. In some embodiments, the cell is a neuron (optionally a dorsal root ganglion neuron or spiral ganglion neuron), astrocyte, cardiomyocyte, or myocyte, astrocyte, glial cell, inner hair cell, outer hair cell, supporting cell, fibrocyte of the inner ear, photoreceptors, interneurons, retinal ganglion, or retinal pigment epithelium.
In some embodiments, the cell is in a living subject, e.g., a mammalian subject, preferably a human. In some embodiments, the cell is in a tissue selected from the brain, spinal cord, dorsal root ganglion, heart, inner ear, eye, or muscle, and a combination thereof. In some embodiments, the subject has Alzheimer's Disease; Parkinson's Disease; X-linked Adrenoleukodystrophy; Canavan's; Niemann Pick; Spinal muscular atrophy; Huntington's Disease; Connexin-26; Usher Type 3A; Usher Type 2D; Hair cell-related hearing loss; Hair cell-related hearing loss (DFNB7/11); Inner hair cell-related hearing loss (DFNB9); Usher Type 1F; Usher Type 1B; Retinitis pigmentosa (RP; non-syndromic); Leber congenital amaurosis; Leber Hereditary Optic Neuropathy; Usher Syndrome (RP; syndromic with deafness); Duchenne Muscular Dystrophy; Allograft vasculopathy; or Hemophilia A and B. The methods and compositions described herein can be used to treat these conditions, 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.
In some embodiments, the cell is in the brain of the subject, and the AAV is administered by parenteral delivery; intracerebral; or intrathecal delivery.
In some embodiments, the intrathecal delivery is via lumbar injection, cisternal magna injection, or intraparenchymal injection.
In some embodiments, the AAV is delivered by parenteral delivery, preferably via intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular delivery.
In some embodiments, the cell is in the eye of the subject, and the AAV is administered by subretinal or intravireal injection.
In some embodiments, the cell is in the inner ear of the subject, and the AAV is administered to 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.
Also provided herein are library construct AAVs comprising:
(i) a sequence encoding a Cre recombinase driven by a promoter;
(ii) a sequence encoding an AAV9 capsid protein with a peptide as described herein, e.g., a heptamer peptide, inserted between the sequences encoding amino acids (aa) 588-589 of the capsid, driven by a promoter, downstream of the Cre cassette. In some embodiments, the peptide comprises a random peptide sequence or a pre-selected peptide sequence.
Further provided are libraries comprising a plurality of the library constructs as described herein. In some embodiments, wherein the peptide sequences are random, the library comprises library constructs having sequences encoding all possible variants of the heptamer.
Additional provided herein are methods for identifying an engineered capsid that mediates transgene expression in a pre-selected cell type. The methods include: (a) administering the library of claim 23 or 24 to a non-human model animal, preferably a mammal, wherein the cells of the model animal express a loxP-flanked STOP cassette upstream of a reporter sequence; (b) isolating cells of the pre-selected cell type; (c) selecting cells in which the reporter sequence is expressed; (d) isolating at least part of the library construct, preferably a part comprising the heptamer, from the selected cells in which the reporter sequence is expressed from step (c); and (e) determining identity of the heptamers in the library constructs isolated in step (d), wherein the heptamers that are isolated can mediate transgene expression in the pre-selected cell type.
In some embodiments, the reporter sequence encodes a fluorescent reporter protein.
In some embodiments, the model animal is transgenic for the loxP-flanked STOP cassette upstream of a reporter sequence, or wherein the loxP-flanked STOP cassette upstream of a reporter sequence can be expressed from a second construct.
In some embodiments, determining identity of the heptamers in the library constructs comprises using DNA sequencing analysis.
In some embodiments, the methods also include before and/or after step (e): using PCR to amplify sequences comprising the heptamer sequences, optionally comprising full capsid sequences, from the library constructs isolated in step (d); cloning the amplified sequences back to a second set of library vectors; repackaging the second set of library vectors; and performing steps (a)-(d) or (a)-(e) on the second set of library vectors.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and Figures, and from the claims.
A promising approach to efficient delivery of transgenes to target cells is via a process of submitting a pool, or library, of AAV vector capsids variants to an in vivo selection process—a veritable “survival of the fittest” approach4-8. AAV library approaches which use random oligomer nucleotides to insert short (6-9 amino acid) random peptides into an exposed region on the capsid surface have demonstrated success in identifying new AAV capsid variants with unique properties such as enhanced transduction of target tissues9, 10. One major limitation of AAV libraries is that the end readout of the selection process does not always differentiate capsids which mediate functional transgene expression from those which do not. AAV transduction is a process involving multiple steps, from cell receptor binding and entry to nuclear transport, second-strand synthesis and finally gene and protein expression11. A recent advance on the conventional AAV library approach, called CREATE, engineered a Cre-sensitive AAV genome which enabled selectively isolate capsids that have successfully trafficked to the nucleus in the context of a Cre-expressing transgenic animal12. Herein is described a capsid selection system, one example of which is called iTransduce, that utilizes the power of the Cre loxP system. Instead of using Cre transgenic mice, the AAV was engineered to encode both the capsids with peptide inserts, along with a Cre-expression cassette. Selection was then performed in mice with a Cre-sensitive fluorescent reporter to enable selection of capsids which mediate the entire process of transduction including transgene expression. In vivo selection of the library resulted in the identification of an AAV capsid that mediates remarkable transduction efficiency of the CNS, and another capsid that mediates transduction in the inner ear.
Using the iTransduce system, we have isolated two new AAV capsids, referred to herein as AAV-F and AAV-S, which mediate highly efficient transgene expression in the murine CNS (two strains tested) and inner ear, respectively. The AAV-F capsid also mediated robust transduction of primary human neurons.
Interestingly, using expression-based selection, 3 peptide clones (STTLYSP (SEQ ID NO:1), FVVGQSY (SEQ ID NO:2), and FQPCP* (SEQ ID NO:3) were identified that represented 97% of NGS reads. Since FQPCP* had a stop codon, it was reasoned that this genome was cross-packaged in another capsid during production, a phenomenon noted to occur with AAV libraries15. This may also be the case for STTLYSP (SEQ ID NO:1, “AAV-S”), (
Since we performed a demonstration of the iTransduce system using an agnostic approach to cell type (whole brain), it is not surprising that AAV-F was highly tropic to astrocytes and neurons, cells that are transduced by AAV9. In future studies we will combine cell specific promoters to drive Cre expression from the AAV library vector as well as magnetic cell sorting to isolate capsids that can transduce cells that are refractory to conventional AAV vector transduction.
In addition to the potential of the iTransduce system to select clinical candidate AAV vectors, it can be used to identify vectors for use as research tools. The recently identified AAV-PHP.B capsid has served as an efficient vector to genetically modify the murine brain12. However, it does not transduce BALB/c or BALB/c related mouse lineages13, 14 16 Interestingly, robust transduction of BALB/c and C57BL/6 murine brain was observed after intravenous injection of AAV-F. This indicates that the mechanism of enhanced transduction over AAV9 differs between AAV-PHP.B and AAV-F. It also enables AAV-F to be utilized as an efficient tool for CNS research in the popular BALB/c strain (labome.com/method/Laboratory-Mice-and-Rats.html). Additionally, as shown herein AAV-F can also mediate robust transgene expression in the CNS after both direct and intrathecal bolus injection, and AAV-S can mediate transgene expression in the inner ear.
Future studies in larger animals can be carried out to further testAAV-F, e.g., in preclinical studies. Dose escalation studies can be performed to test for dose-related toxicity of AAV-F, as was observed with PHP.B in NHPs.14. Iterative rounds of selection can be done in different species (e.g. mice then rats) to allow better cross-species translation of the transduction efficiency. For example, this could be done in mice followed by floxed STOP tdTomato transgenic rats17. Alternatively, direct selection of the iTransduce library can be performed in transgenic marmosets18, 19 or even in other non-human non-transgenic primates (
Methods of Identifying Optimized Capsid Sequences
“Virus vector libraries” are pooled variants of viruses, which under selective pressure (in vivo or in vitro) can drive isolation of clones of viruses specific for a target cell/tissue/organ of interest. One limitation of current library technologies is that many of the candidate virus clones do not mediate transgene expression (the required final function of the vector). The main reason for this limitation is that there has been no strategy devised to allow vector selection based on vector-mediated transgene expression. Described herein are methods that use an adeno-associated virus (AAV) vector genome with a two-part expression cassette. The first is a Cre-recombinase cassette under a promoter of interest. The second part is an AAV promoter to drive expression of the capsid gene, cloned “in cis” to the first section of the viral genome. Virus vectors can now be selected for transgene expression (highly sensitive Cre expression) using cells that express a reporter gene (e.g., green fluorescent protein) with an upstream loxP/stop site, thus preventing reporter expression until AAV vector-delivered Cre removes the stop site. Reporter gene positive cells can be isolated and recovered AAV capsid sequences will have a higher likelihood of mediating efficient transgene expression.
Thus provided herein are library construct AAVs comprising: (i) a Cre recombinase driven by a promoter, e.g., a minimal chicken beta actin (CBA) promoter; (ii) a promoter (e.g., p41 promoter)-driven AAV9 capsid sequence with a sequence encoding a peptide as described herein, e.g., a random heptamer peptide or selected heptamer peptide, inserted into a capsid protein, downstream of the Cre cassette. Preferably the peptide is inserted between the sequences encoding amino acids (aa) 588-589 of the capsid, but it can also be inserted elsewhere as long as it doesn't interfere with function of the virus and maintains its activity in promoting infection of selected cells, e.g., at the VP1/VP2 interface (amino acid 138) or any site between 583-590. The CBA promoter is strong, active promoter to drive Cre in most cell types. The P41 promoter is an AAV specific natural promoter which drives Cap gene expression Other promoters that can be used include, but are not limited to, Synapsin promoter, GFAP promoter, CD68 promoter, F4/80 promoter, CX3CR1 promoter, CD3 or CD4 promoter, CMV promoter, liver specific promoter; other examples are listed below. The constructs can also include a stop codon at the end of the Cre cDNA and at the end of the cap DNA. There are poly A signals after the Cre cassette and the cap cassette. Cre recombinases are known in the art, see, e.g., Van Duyne, Microbiol Spectr. 2015 February; 3(1):MDNA3-0014-2014.
Also provided herein are libraries (i.e., compositions comprising a plurality of the library constructs). Where random heptamer sequences are used, preferably the library comprises constructs with sequences encoding all or almost all possible variants of the heptamer).
The methods, illustrated in
Promoters
The library constructs described herein include two promoters; one driving the Cre recombinase, and a second driving the AAV capsid sequence.
A number of promoter sequences are known in the art, including so-called “ubiquitous” promoters that drive expression in most cell types, e.g., cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), chicken beta-actin (CBA) promoter, Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), SV40 promoter, dihydrofolate reductase promoter, phosphoglycerol kinase promoter, phosphoglycerol kinase (PGK) promoter, EFlalpha promoter, Ubiquitin C (UBC), B-glucuronidase (GUSB), and CMV immediate/early gene enhancer/CBA promoter.
Expression of the Cre-recombinase can also be driven by a tissue-specific promoter, e.g., a tissue-specific promoter for CNS, liver, heart cochlea, retina, or T cells, inter alia. In some embodiments, the tissue specific promoter for CNS includes neuronal, macrophage/microglial promoter and astrocyte promoters. A number of tissue specific promoters are known in the art, including synapsin promoter (neurons), neuron-specific enolase (NSE) (neurons), MeCP2 (methyl-CPG binding protein 2) (neurons), a glial fibrillary acidic protein (GFAP) (astrocytes), oligodendrocyte transcription factor 1 (Olig1) (oligodendrocytes), CNP (2′,3′-Cyclic-nucleotide 3′-phosphodiesterase) (broad), or CBh (hybrid CBA or a MVM intron with CBA promoter)(broad). See, e.g., US20190032078. Macrophage/microglial promoters include, but are not limited to, a C-X3-C motif chemokine receptor 1 (CX3CR1) promoter, CD68 promoter, an ionized calcium binding adaptor molecule 1 (IBA1) promoter, a transmembrane protein 119 (TMEM119) promoter, a spalt like transcription factor 1 (SALL1) promoter, an adhesion G protein-coupled receptor E1 (F4/80) promoter, a myeloproliferative sarcoma virus enhancer, negative control region deleted, d1587rev primer-binding site substituted (MND) promoter; integrin subunit alpha M (ITGAM; CD11b-myeloid cells (neutrophils, monocytes, and macrophages)) promoter. For expression in the inner ear, the promoter can be, e.g., a PKG, CAG, prestin, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, oncomod, or myo7A promoter; see Ryan et al., Adv Otorhinolaryngol. 2009; 66: 99-115.
Reporter Proteins
A number of reporter proteins are known in the art, and include green fluorescent protein (GFP), variant of green fluorescent protein (GFP10), enhanced GFP (eGFP), TurboGFP, GFPS65T, TagGFP2, mUKGEmerald GFP, Superfolder GFP, GFPuv, destabilised EGFP (dEGFP), Azami Green, mWasabi, Clover, mClover3, mNeonGreen, NowGFP, Sapphire, T-Sapphire, mAmetrine, photoactivatable GFP (PA-GFP), Kaede, Kikume, mKikGR, tdEos, Dendra2, mEosFP2, Dronpa, blue fluorescent protein (BFP), eBFP2, azurite BFP, mTagBFP, mKalamal, mTagBFP2, shBFP, cyan fluorescent protein (CFP), eCFP, Cerulian CFP, SCFP3A, destabilised ECFP (dECFP), CyPet, mTurquoise, mTurquoise2, mTFPI, photoswitchable CFP2 (PS-CFP2), TagCFP, mTFP1, mMidoriishi-Cyan, aquamarine, mKeima, mBeRFP, LSS-mKate2, LSS-mKatel, LSS-mOrange, CyOFP1, Sandercyanin, red fluorescent protein (RFP), eRFP, mRaspberry, mRuby, mApple, mCardinal, mStable, mMaroonl, mGarnet2, tdTomato, mTangerine, mStrawberry, TagRFP, TagRFP657, TagRFP675, mKate2, HcRed, t-HcRed, HcRed-Tandem, mPlum, mNeptune, NirFP, Kindling, far red fluorescent protein, yellow fluorescent protein (YFP), eYFP, destabilised EYFP (dEYFP), TagYFP, Topaz, Venus, SYFP2, mCherry, PA-mCherry, Citrine, mCitrine, Ypet, IANRFP-AS83, mPapayal, mCyRFP1, mHoneydew, mBanana, mOrange, Kusabira Orange, Kusabira Orange 2, mKusabira Orange, mOrange 2, mKOK, mKO2, mGrapel, mGrape2, zsYellow, eqFP611, Sirius, Sandercyanin, shBFP-N158S/L173I, near infrared proteins, iFP1.4, iRFP713, iRFP670, iRFP682, iRFP702, iRFP720, iFP2.0, mIFP, TDsmURFP, miRFP670, Brilliant Violet (BV) 421, BV 605, BV 510, BV 711, BV786, PerCP, PerCP/Cy5.5, DsRed, DsRed2, mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, or a Phycobiliprotein, or a biologically active variant or fragment of any one thereof.
Kits
Also provided herein are kits comprising one or more library construct AAVs as described herein, with or without the random heptamer sequences. The kits can also include a construct comprising a loxP-flanked STOP cassette upstream of a reporter sequence.
Engineered AAV Capsid Proteins
The present methods identified two peptide sequences that alter the ability of an AAV to mediate transgene expression in specified cells when inserted into the capsid of the AAV, e.g., AAV1, AAV2, AAV8, or AAV9. In some embodiments, the peptides comprise sequences of at least 7 amino acids. In some embodiments, the amino acid sequence comprises at least 4, e.g., 5, 6, or 7 contiguous amino acids of the sequences (STTLYSP (SEQ ID NO: 1) or FVVGQSY (SEQ ID NO:2).
Peptides including reversed sequences can also be used, e.g., PSYLTTS (SEQ ID NO:4) and YSQGVVF (SEQ ID NO:5). Alternatively, the peptides can comprise at least four, five, or six contiguous amino acids from the sequences shown in
A AVs
Viral vectors for use in the present methods, kits and compositions include recombinant retroviruses, adenovirus, adeno-associated virus, alphavirus, and lentivirus, preferably comprising a capsid peptide as described herein and optionally a transgene for expression in a target tissue.
A preferred viral vector system useful for delivery of nucleic acids in the present methods is the adeno-associated virus (AAV). 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. 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). There are numerous alternative AAV variants (over 100 have been cloned), and AAV variants have been identified based on desirable characteristics. In some embodiments, 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. Using the present methods, the AAV capsid can be genetically engineered to increase permeation across the BBB, or into a specific tissue, 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:
Thus provided herein are 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., a capsid protein comprising SEQ ID NO:1 or SEQ ID NO:2, wherein a peptide sequence has been inserted into the sequence, e.g., between amino acids 588 and 589.
Exemplary sequences of AAVs are provided below. The inserted peptide sequences are bold and double-underlined highlighted in the protein sequences, and bold and capitalized in the DNA sequences.
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. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, 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). In a preferred embodiment, 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. For example, 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.
Transgenes
In some embodiments, 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. The transgene is preferably linked to sequences that promote/drive expression of the transgene in the target tissue.
Exemplary 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 (ASPA), 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), bone morphogenetic protein (BMP), epidermal growth factor (EGF), growth differentiation factor-9 (GDF-9), hepatoma derived growth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), and the like; soluble receptors, such as soluble TNF-α receptors, soluble VEGF receptors, soluble interleukin receptors (e.g., soluble IL-1 receptors and soluble type II IL-1 receptors), soluble gamma/delta T cell receptors, ligand-binding fragments of a soluble receptor, and the like; enzymes, such as a-glucosidase, imiglucarase, and P-glucocerebrosidase; enzyme activators, such as tissue plasminogen activator; chemokines, such as IP-10, monokine induced by interferon-gamma (Mig), Groa/IL-8, RANTES, MIP-la, MIP-10, MCP-1, PF-4, and the like; angiogenic agents, such as vascular endothelial growth factors (VEGFs, e.g., VEGF121, VEGF165, VEGF-C, VEGF-2), transforming growth factor-beta, basic fibroblast growth factor, glioma-derived growth factor, angiogenin, angiogenin-2, and the like; anti-angiogenic agents, such as a soluble VEGF receptor; protein vaccine; neuroactive peptides, such as nerve growth factor (NGF), bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagons, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleep peptide, and the like; thrombolytic agents; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle stimulating hormone (FSH); human alpha-1 antitrypsin; leukemia inhibitory factor (LIF); transforming growth factors (TGFs); tissue factors, luteinizing hormone; macrophage activating factors; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); nerve growth factor; tissue inhibitors of metalloproteinases; vasoactive intestinal peptide; angiogenin; angiotropin; fibrin; hirudin; IL-1 receptor antagonists; and the like. Some other examples of protein of interest 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, β-enolase, and glycogen synthase; lysosomal enzymes (e.g., beta-N-acetylhexosaminidase A); and any variants thereof.
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; CD152); 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).
Other 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), or CRISPR Cas9/cas12a and guide RNAs.
The virus 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. In some embodiments, the promoter is a brain tissue specific promoter, e.g. a neuron-specific or glia-specific promoter. In certain embodiments, 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. In some embodiments, the promoter is a pan-cell type promoter, e.g., cytomegalovirus (CMV), beta glucuronidase, (GUSB), ubiquitin C (UBC), or rous sarcoma virus (RSV) promoter. The woodchuck hepatitis virus posttranscriptional response element (WPRE) can also be used.
In some embodiments, 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 Pulicherla et al. (2011) Mol Ther 19:1070-1078); or the addition of other peptides, e.g., as described in Chen et al. (2008) Nat Med 15:1215-1218 or Xu et al., (2005) Virology 341:203-214 or U.S. Pat. Nos. 9,102,949; 9,585,971; and US20170166926. See also Gray and Samulski (2011) “Vector design and considerations for CNS applications,” in Gene Vector Design and Application to Treat Nervous System Disorders ed. Glorioso J., editor. (Washington, D.C.: Society for Neuroscience; ) 1-9, available at sfn.org/˜/media/SfN/Documents/Short %20Courses/2011%20Short %20Course %20I/2011_SC1_Gray.ashx.
Methods of Use
The methods and compositions described herein can be used to deliver any composition, e.g., a sequence of interest to a tissue, e.g., to the central nervous system (brain), heart, muscle, peripheral nervous system (e.g., dorsal root ganglion or spinal cord), or to the inner ear or retina. In some embodiments, the methods include delivery to specific brain regions, e.g., cortex, cerebellum, hippocampus, substantia nigra, amygdala. In some embodiments, the methods include lumbar delivery, e.g., into the subarachnoid space or epidural space. In some embodiments, the methods include delivery to neurons, astrocytes, or glial cells. In some embodiments, the methods include delivery to inner and/or outer hair cells, spiral ganglion neurons, supporting cells, or fibrocytes of the inner ear. In some embodiments, the methods include delivery to the photoreceptors, interneurons, retinal ganglion cells (e.g., using AAV-F), or retinal pigment epithelium (RPE) (e.g., using AAV-S) of the retina.
In some embodiments, the methods and compositions, e.g., AAVs, 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., U.S. Pat. Nos. 9,102,949; 9,585,971; and US20170166926. In some embodiments, the subject has a condition listed in Tables 1-3; in some embodiments, the vectors are used to deliver a therapeutic agent listed in Tables 1-3 for treating the corresponding disease listed in Tables 1-3. 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 other nucleic acid such as an antisense oligo, siRNA, shRNA, and so on; or as a fusion protein/complex with a peptide as described herein.
The methods and 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.
Pharmaceutical Compositions and Methods of Administration
The methods described herein include the use of pharmaceutical compositions comprising the AAVs as an active ingredient.
Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language “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.
Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of 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. For example, for delivery into the inner ear, 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. 2019 Jan. 11; 13:197-204; Ren et al., Front Cell Neurosci. 2019; 13: 323); for delivery into the retina, subretinal or intravitreal injections can be used (see, e.g., Ochakovski et al., Front Neurosci. 2017; 11: 174; Xue et al., Eye (Lond). 2017 September; 31(9):1308-1316).
Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, N.Y.). For example, 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.
Pharmaceutical 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. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, 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. In many cases, it will be preferable to include 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. Generally, 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. In the case of sterile powders for the preparation of sterile injectable solutions, 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.
In one embodiment, 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. 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. Pat. No. 4,522,811.
The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration. For example, the kit can include compositions comprising an AAV comprising a peptide as described herein.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Materials and Methods
The following materials and methods were used in the Examples below, unless otherwise noted.
AAV Library Construction.
iTransduce Plasmid: pAAV-CBA-Cre-mut-p41-Cap9del
We constructed the iTransduce library backbone plasmid called pAAV-CBA-Cremut-p41-Cap9del, containing two expression cassettes in-cis: 1) the CBA-Cremut in which we introduced a mutant Cre cDNA (CCG->CCT encoding the Pro15 amino acid to eliminate the AgeI site initially present) under the ubiquitous promoter CBA, 2) the p41-Cap9del composed of the AAV9 capsid gene under the AAV5 p41 promoter (residues 1680-1974 of GenBank AF085716.1) and splicing sequences of the AAV2 rep gene (similar as described in12).
Both the mutant Cre cDNA (Cremut) flanked by KpnI and SalI restriction sites and the p41-CAP9(del)-polyA fragment were synthesized by GenScript and cloned into a puC57 backbone. We constructed first the pAAV-CBA-Cremut-polyA plasmid by subcloning the mutant Cre cDNA (fragment KpnI-blunt, SalI) in place of eGFP-WPRE in our pAAV-CBA-WPRE backbone (opened with NcoI-blunt/SalI, which eliminated the eGFP-WPRE fragment initially present). We then introduced the p41-Cap9del fragment harbouring a K449R mutation in the Cap9 sequence allowing the creation of a unique XbaI site, and in which the 447 bp Cap sequence between XbaI and AgeI restriction sites had been deleted.
Plasmid for Generating Random 7-Mer Peptide Cap Fragments and Subcloning the CAP9 Fragments Retrieved by PCR: pUC57-Cap9-XbaI/KpnI/AgeI.
We first had the 447 bp region of the AAV9 capsid sequence between the XbaI/AgeI sites (sequence that is missing in pAAV-CBA-Cremu t-p41-Cap9del) synthesized by Genscript (Piscataway, N.J.) and subcloned into a pUC57-Kan plasmid. This capsid sequence contains the same removal of the Earl site in this 447 bp region WT AAV9 cap that allows restriction digest removal of WT AAV9 as described by Deverman et al. 2015. This also creates a unique KpnI site. The cap fragment of pUC57-Cap9-XbaI/KpnI/AgeI was used to generate the initial library of random 21-mer nucleotide sequences (encoding for 7-mer peptides) inserted between nucleotides encoding amino acids 588 and 589 of AAV9 VP1. We used a strategy similar to that of Deverman et al. (2015). Briefly, pUC57-Cap9-XbaI/KpnI/AgeI served as template to amplify the cap DNA and insert random 21-mer sequences using a forward and reverse primer. Primer information: XF-extend (5′GTACTATCTCTCTAGAACtattaacggttc3′; SEQ ID NO: 11) and reverse primer 588iRev 5′ (GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCXMNNMNNMNNM NNMNNMNNMNNTTGGGCACTCTGGTGGTTTGTG 3′; SEQ ID NO: 12) in which the MNN repeat refers to the the randomized 21-mer nucleotides (purchased from IDT). XF-extend and 588iRev were used in a PCR with Phusion polymerase (NEB) and pUC57-Cap9-XbaI/KpnI/AgeI as template. The 447 bp PCR product was digested with XbaI and AgeI overnight at 37° C. and then gel-purified the product (Qiagen). Similarly, pAAV-CBA-Cre-mutp41-Cap9del was digested with XbaI and AgeI and gel purified. Next, a ligation reaction (1 h at room temperature) with T4 DNA ligase (NEB) was performed using a 3:1 cap insert to vector molar ratio. The subsequent ligated plasmid was called pAA V-CBA-Cre-mut-p41-Cap9-7mer and contained a pool of plasmids with random 7-mer peptides inserted in the cap gene between nucleotides encoding 588 and 589 of AAV9 VP1.
This plasmid (pUC57-Cap9-XbaI/KpnI/AgeI) was also used as our recipient plasmid for subcloning the CAP9 fragments amplified by PCR from brain tissue. We removed an upstream KpnI site in the pUC57 plasmid by digestion with SacI and NsiI and ligation. This allowed the KpnI site in the capsid fragment to be unique. See below in Rep expression plasmid.
We constructed a rep expression plasmid called pAR9-Cap9-stop/AAP/Rep using a similar strategy to that as Deverman et al.12. The entire cDNA was synthesized by Genscript and cloned into a pUC57-Kan plasmid. Stop codons were inserted in place of start codons for VP1, VP2, VP3 so that no parental AAV9 capsids were produced, while maintaining AAP and rep expression.
AAV Library Production and Purification.
For each production, we plated 15-cm tissue culture dishes with 1.5×107 293T cells/dish. The next day cells were transfected using the calcium phosphate method, with the adenovirus helper plasmid (pAdΔF6, 26 μg per plate), rep plasmid (pAR9-Cap9-stop/AAP/Rep, 12 μg per plate) and ITR-flanked AAV library (pAAV-CBA-Cre-mut/p41-Cap9-7mer, 1 μg per plate) to induce production of AAV. The day after transfection, medium was changed to DMEM containing 2% FBS. AAV was purified from the cell lysate using iodixanol density-gradient ultracentrifugation. Buffer exchange to PBS was done using ZEBA spin columns (7K MWCO; Thermo Fisher Scientific) and further concentration was performed using Amicon Ultra 100 kDa MWCO ultrafiltration centrifugal devices (Millipore). Vectors were stored at −80° C. until use. We quantified AAV genomic copies (vg) in AAV preparations using TaqMan qPCR with ITR-sequence specific primers and probes20, 21.
Next Generation Sequencing of Library.
Next generation sequencing was performed on the plasmid AAV9 library pool, as well as following packaging of capsids. Sequencing was also performed following PCR rescue of the cap fragment (either from brain tissue or from isolated tdTomato-positive cells sorted by flow cytometry). For each round of selection viral DNA corresponding to the insert-containing region was amplified by PCR using the Phusion High-Fidelity PCR kit from New England Biolabs (Forward primer: 5′-AATCCTGGACCTGCTATGGC-3′ (SEQ ID NO:13), reverse primer: 5′-TGCCAAACCATACCCGGAAG-3′ (SEQ ID NO:14)). PCR amplification was performed using Q5 polymerase (New England Biolabs). Unique barcode adapters were annealed to each sample, and samples were sequenced on an Illumina Miseq (150 bp reads). Approximately 50-100,000 reads per sample were analysed. 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.
Animals.
All animal experiments were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care following guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. We used adult age (8-10 week old) Ai9 (strain #007909), C57BL/6 (strain #000664), and BALB/c (strain #000651) mice all from The Jackson Laboratory, Bar Harbor, Me. All animals were euthanized three weeks post-injection, perfused transcardially and tissues were harvested and either fixed in 4% paraformaldehyde in PBS, snap frozen in liquid nitrogen or dissociated for flow cytometry.
In Vivo Selection of Brain-Tropic Capsids.
Ai9 mice were injected intravenously (tail vein) with the dose in vg indicated in the results section and 3 weeks post injection, mice were euthanized, and tissue harvested.
Mice were deeply anesthetized by isofluorane and decapitated. For round 1, the brain was rapidly dissected and two coronal sections (2 mm thick) were harvested. One section was used for extracting whole brain DNA (DNeasy Blood and Tissue Kits, Qiagen, Hilden, Germany). The other coronal section was fixed in 4% PFA and paraffin embedded for immunohistology (tdtomato-positive cells were detected after each round of selection by DAB staining using a rabbit anti-RFP antibody from Rockland Immunochemicals). For round 2, brain tissue was then cut with a razor blade into 1 mm3 pieces and neural cells were isolated by papain dissociation (Papain Dissociation System, Worthington), according to the manufacturer's instructions. Following dissociation, myelin was removed (Myelin Removal Beads II, human, mouse, rat from Miltenyi) and the Td-tomato positive cells were sorted by a S3eTM Cell Sorter (Bio-Rad). Cells were sorted by first setting gates to exclude cellular debris and select for singlets only. Cell suspensions from an AAV9-PHP.B-Cre injected Ai9 (positive control) and a PBS injected Ai9 mouse (negative control) were used to set gates to sort tdTomato-positive and negative cells. After sorting, the tdTomato-positive cells were immediately pelleted by centrifugation, and DNA was extracted using the ARCTURUS PicoPure DNA extraction kit (ThermoFisher).
After DNA extraction, the Cap9 inserts (containing the 21-mer sequence encoding the 7mer peptides) were amplified using the following primers: Cap9_Kpn/Age_For: 5′-AGCTACCGACAACAACGTGT-3′ (SEQ ID NO:15) and Cap9_Kpn/Age_Rev: 5′-AGAAGGGTGAAAGTTGCCGT-3′ (SEQ ID NO:16) (Phusion High-Fidelity PCR kit, New England Biolabs). The amplicons were then purified (Monarch PCR & DNA Cleanup kit, New England Biolabs), digested by KpnI, AgeI and BanII and the Cap9 KpnI-AgeI fragments (144 bp) were agarose gel purified (Monarch DNA Gel Extraction kit, New England Biolabs) before ligation in the pUC57-Cap9-XbaI/AgeI/KpnI plasmid (opened with KpnI and AgeI and dephosphorylated with Calf Inositol Phosphatase, New England Biolabs). The ligation products were transformed into electrocompetent DHSalpha bacteria (New England Biolabs) and the entire transformation was grown overnight in LB-ampicillin medium. pUC57-Cap9-XbaI/AgeI/KpnI plasmid was purified by maxi prep (Qiagen). Plasmid was digested by XbaI/AgeI to release the 447 bp cap fragment which was gel purified and ligated with similarly cut pAAV-CBA-Cre-mut/p41-Cap9del for the next round of AAV library production.
AAV Rep Cap Plasmids Containing AAV-F and AAV-S Peptide Inserts for Vector Production.
To create rep cap plasmids encoding AAV9 capsids displaying the peptide insert of interest for production of vectors encoding a transgene of interest (e.g. GFP), we digested an AAV9 rep cap plasmid with BsiWI and BaeI which removes a fragment flanking the VP3 amino acid 588 site for peptide sequence insertion. Next we ordered a 997 bp dsDNA fragment from Integrated DNA Technologies (IDT, Coralville, Iowa), which contains overlapping Gibson homology arms with the BsiWI/BaeI cut AAV9 as well as the 21-mer nucleotide sequence encoding the peptide of interest in frame after amino acid 588 of VP3. Last, we performed Gibson assembly using the Gibson Assembly® Master Mix (NEB, Ipswich, Mass.) to ligate the peptide containing insert into the AAV9 rep cap plasmid.
AAV Vectors for Transduction Analysis.
For each production, we plated 15-cm tissue culture dishes with 1.5×107 293T cells/dish. The next day cells were transfected using the calcium phosphate method, with the adenovirus helper plasmid (pAdΔF6, 26 μg per plate), rep/cap plasmid (AAV9, AAV-F, AAV-S; 12 μg per plate) and ITR-flanked transgene cassette plasmid (single-stranded AAV-CBA-GFP-WPRE22, 10 μg/plate) to induce production of AAV. The day after transfection, medium was changed to DMEM containing 2% FBS. AAV was purified from the cell lysate using iodixanol density-gradient ultracentrifugation. Buffer exchange to PBS was done using ZEBA spin columns (7K MWCO; Thermo Fisher Scientific) and further concentration was performed using Amicon Ultra 100 kDa MWCO ultrafiltration centrifugal devices (Millipore). Vectors were stored at −80° C. until use. We quantified AAV genomic copies in AAV preparations using TaqMan qPCR with BGH polyA-sequence specific primers and probe23.
Animal Euthanasia and Tissue Harvesting
Mice (strain indicated in each FIGure) were slowly injected via the lateral tail vein with 200 μl of the tested AAV vector diluted in sterile PBS (low dose: 4×1012 vg/kg and high dose: 3.2×1013 vg/kg), before gently finger-clamping the injection site until bleeding stopped. Three weeks post injection mice were euthanized and perfused transcardially with sterile cold phosphate buffered saline (PBS). Next the brain was longitudinally bisected into two hemispheres. One hemisphere was post-fixed in 15% Glycerol/4% paraformaldehyde diluted in PBS for 48 hours, followed by 30% glycerol for cryopreservation for another 48-72 hours. For the high-dose cohort, a small piece of heart, muscle (gastrocnemius) and the retina were also processed for immunohistology. We made 3 independent preparations of AAV-S, AAV-F, and AAV9 (Table I). The transduction results in mice were from one preparation of each vector, however we have replicated these results in two more independent experiments.
Immunohistology and High-Magnification Imaging of AAV-CBA-GFP Transduced Neural and Neuronal Cell Populations
Coronal floating sections (40 μm) were cut using a cryostat microtome. After rinsing off the glycerol in tris-buffered saline (TBS) buffer, cryosections were permeabilized with 0.5% Triton X-100 (AmericanBio) in TBS for 30 minutes at room temperature and blocked with 5% normal goat serum (or normal donkey serum) and 0.05% Triton in TBS for 1 hour at room temperature. Primary antibodies were incubated overnight at 4° C. in 2.5% NGS and 0.05% Triton in TBS, while Alexa Fluor 488 or -Cy3 conjugated secondary antibodies (Jackson ImmunoResearch laboratories, Baltimore, USA) were incubated for 1 hour the next day. Primary antibodies used for this study were: chicken anti-GFP (Aves Labs, Tigard, USA), Mouse anti-NeuN (EMD Millipore, Burlington, USA); rabbit anti-Glutamine Synthetase (Abcam, Cambridge, USA); rabbit anti-Olig2 (EMD Millipore, Burlington, USA); rabbit anti-Iba1 (Wako, Japan); rabbit anti-CamKII (Abcam, Cambridge, USA); mouse anti-GAD67 (EMD Millipore, Burlington, USA); rabbit anti-ChAT (EMD Millipore, Burlington, USA); mouse anti-calbindin (Abcam, Cambridge, USA) and rabbit anti-TH (Novus Biologicals, Littleton, USA). Sections were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, USA).
To identify the neural cell types transduced by each vector and investigate the various neuronal sub-types targeted by AAV-F, a Zeiss Axio Imager Z epifluorescence microscope equipped with AxioVision software and a 60× objective was used to take high-resolution images showing colocalization between GFP and each cell marker.
Imaging and Quantification of Global GFP Signal Coverage
To quantify the overall native GFP fluorescence signal in brain and liver section, a robotic slide scanner Virtual slide microscope VS120 (Olympus) was used to image the entire batch of slides on one go using an Olympus UPLSAPO 10× objective. In order to reduce variability, the entire batch of slides was imaged in one session. The initial exposure time for GFP was set up so that the fluorescent signal was neither under-no over-saturated across all experimental group and remained unchanged throughout the entire batch scan. The order of the slides was randomized and remained blinded until final statistical analysis. The Olympus cellSens Standard software was then used to analyze the percent GFP coverage in each brain section. A region of interest (ROI) was initially defined using the “ROI-polygon” tool and we quantified the GFP-positive area within this initial ROI, after applying a similar detection threshold on the GFP channel for all the slides analyzed (the threshold was set at a similar level for the analysis of all mouse brain sections, but a different threshold was applied for the analysis of all mouse liver sections and all rat brain sections). The percentage of GFP-positive area accordingly to the total surface of the ROI was then calculated. The autofluorescence signal was taken into account in our analysis as we set the threshold for eGFP fluorescence intensity above the autofluorescence level (making sure that only the signal from AAV-GFP transduced cells was taken into account). In addition, we drew each ROI for each brain section avoiding the very edges of the section, as those could also present with a high level of autofluorescence. The ventricular space was also excluded from our analysis. Finally, three technical replicates (brain sections) were measured per mouse and all measurements were done blind until the final step of the analysis.
Stereology-Based Quantitative Analyses of the Percentages of Transduced Astrocytes and Neurons
Stereology-based studies were performed as previously described24, 25, after co-staining the brain sections for GFP and NeuN (neuronal marker) or GFP and GS (Glutamine synthetase, pan-astrocytic marker). We did not include microglia and oligodendrocytes in this analysis as those cell types were not transduced to an appreciable amount. Stereological evaluation of the percentages of AAV-transduced neurons and astrocytes was done blindly after de-identification of the vector initially injected, using a motorized stage of an Olympus BX51 epifluorescence microscope equipped with a DP70 digital CCD camera, an X-Cite fluorescent lamp, and the associated CAST stereology software version 2.3.1.5 (Olympus, Tokyo, Japan). The cortex was initially outlined under the 4× objective. Random sampling of the selected area was defined using the optical dissector probe of the CAST software. To evaluate the percentage of AAV9, AAV9-PHP.B, AAV-S and AAV-F transduced astrocytes or neurons, the stereology-based counts were performed under the 20× objective, with a meander sampling of 10% for the surface of cortex for the “high transduction” AAVs, and 20% for “low transduction” AAVs (considering the infrequency of GFP positive cells in those cases ). For each counting frame, the total number of astrocytes (GS positive cells) or neurons (NeuN positive cells) were evaluated, and, among each of those populations, the percentages of GFP positive cells. Only glial and neuronal cells with DAPI-positive nucleus within the counting frame were considered.
Vector Genome Quantification in the Brain and Liver
One brain hemisphere and a small piece of liver were fresh frozen for AAV genome isolation for vector genome biodistribution. For the fresh frozen brain and liver samples, we isolated genomic and AAV vector DNA from 10 mg of tissue using the DNeasy Blood and Tissue Kit (Qiagen) according to manufacturer's instructions. DNA was quantitated using a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). Next using 50 ng of genomic DNA as template, we performed a Tagman qPCR using probe and primers to the polyA region of the transgene expression cassette (same assay used to titer the purified AAV vectors). To ensure equal genomic DNA input for each sample, we performed a separate qPCR on each sample using a Tagman probe and primer set that detects GAPDH genomic DNA (Thermo Fisher Scientific, Assay ID Mm01180221_g1; gene symbol Gm12070). For each organ/tissue, we adjusted the AAV vector genome copies for each sample by taking into account any differences in GAPDH Ct values using the following formula: (AAV vector genome copies)/(2ΔCt). The ΔCt value was calculated by the following formula: GAPDH Ct value of sample of interest−average GAPDH Ct value of sample which had the lowest amount of GAPDH (highest Ct value). Data was expressed as AAV vector genomes per 50 ng genomic DNA.
Human Neuron Transduction:
Primary human fetal neural stem cells (NSCs) were obtained from the Birth Defects Research Laboratory (University of Washington, Seattle, Wash.) in full compliance with the NIH ethical guidelines. The isolation procedure has been detailed previously26 with slight modifications. Briefly, brain tissue was incubated in 0.25% trypsin, DNase (90 Units/mL), diluted in Hank's balanced salt solution (HBSS) for 45 min. Tissue was titrated, transferred into 4° C. heat-inactivated fetal bovine serum and centrifuged at 500 g for 20 min. The pellet was resuspended in NSC complete media consisting of x-Vivo 15 (without phenol red and gentamicin; Lonza) supplemented with 10 μg of basic fibroblast growth factor (Life Technologies), 100 μg of epidermal growth factor (Life Technologies), 5 μg of leukemia inhibitory factor (EMD Millipore), 60 ng/mL of N-acetylcysteine (Sigma-Aldrich), 4 mL of neural survival factor-1 supplement (Lonza), 5 mL of 100× N-2 supplement (Life Technologies), 100 U of penicillin, 100 μg/mL of streptomycin (Life Technologies), and 2.5 μg/mL of fungizone (Life Technologies). Supernatants were then filtered through a 40 μm cell strainer (Corning Life Science). Neurospheres larger than 40 m in diameter were dissociated with Accutase (10 min). Neural Differentiation Medium consisted of 1× Neurobasal Medium, 2% B-27 serum-free supplement, and 2 mM GlutaMAX-I supplement (all from Invitrogen) and supplemented with human recombinant brain-derived neurotrophic factor (BDNF) (10 ng/mL; Peprotech).
Differentiating NSCs were grown in chamber slides in differentiation media for 2 weeks and then treated with the indicated AAV vector encoding GFP (7×109 vg/well added, 150 vg/cell). One week after transduction, cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% Triton X-100 (Sigma-Aldrich) in 1× phosphate-buffered saline (PBS; Invitrogen). Cells were stained with a primary monoclonal antibody (TU-20) to neuron-specific class III P-Tubulin (1:50; Abcam). Secondary antibodies conjugated to Alexa Fluor 594 (diluted 1:200; Invitrogen) were added for 1 h, followed by DAPI for 30 min. The slides were then mounted with a ProLong antifade reagent (Invitrogen). Max projection Images were generated form captured Z-stacks using the Nikon AiR confocal microscope.
Z-stacks were loaded in Imaris, the surface module was used to render the images into 3D volumes. GFP+ neurons were counted (under channel 1-green) and Class III β-Tubulin positive neurons (under channel 2-red). Using Imaris' colocalization module, the population of neurons double positive for the above was determined.
Statistics
Statistical analysis of data was performed using GraphPad Prism software (version 8.00). A one-way ANOVA test followed by a Tukey's multiple comparisons test was performed, across the different groups AAV9, AAV9-PHP.B, AAV-S and AAV-F. A value of p<0.05 was considered to be statistically significant. Results are shown as the mean±S.E.M. Similar analyses were performed the biodistribution assay of AAV genomes in brain and liver, as well as transduction of human neurons.
Direct Stereotactic Injection of Vectors
Adult C57BL/6 mice were anesthetized by intraperitoneal injection of ketamine/xylazine (100 mg/kg and 50 mg/kg body weight, respectively) and positioned on a stereotactic frame (Kopf Instruments, Tujunga, USA). Injections of vectors were performed in the cortex (somatosensory cortex) and the hippocampus. A total of 3 μl of viral suspension was injected (1.65×1010 and 5.6×1010 gc per injection site for AAV-F and AAV-S, respectively) at a rate of 0.15 μl/minute) and using a 33-gauge sharp needle attached to a 10-μl Hamilton syringe (Sigma-Aldrich, St. Louis, USA). Stereotactic coordinates of injection sites were calculated from bregma (Cortex coordinates: anteroposterior −1 mm, mediolateral±1 mm and dorsoventral −0.8 mm; Hippocampus coordinates: anteroposterior −2 mm, mediolateral±1.7 mm and dorsoventral −2.5 mm).
Intrathecal Bolus Delivery (IT Bolus)
Adult C57BL/6 mice were put under anesthesia by isoflurane. After the skin over the lumbar region was shaved and cleaned, a 3˜4 cm mid-sagittal incision was made through the skin exposing the muscle and spine. A catheter was inserted between L4-L5 spine region and attached to a gas-tight Hamilton syringe with a 33-gauge steel needle. Ten microliters of AAV9-CBA-GFP (1.25×1011 vg) vectors or AAV-F-CBA-GFP (8.8×1010 vg) were slowly injected at a rate of 2 μl/min. Mice were killed three weeks post injection.
For low magnification imaging of whole spinal cord and brain sections, we stained overnight with anti-GFP (Invitrogen, cat no. G10362, dilution 1:250) followed by secondary antibody staining and imaging as for
For immunostaining of cell types in brain and spinal cord, fixed tissue sections were washed with PBS, blocked with goat serum and permeabilized with 0.3% Triton in PBS. Target antibodies were diluted in blocking buffer and incubated at 4° C. overnight.
Primary antibodies: anti-GFP: catalog no. ab1218 (abcam): Dilution, 1:1000
After overnight incubation slides were washed extensively with PBS with 0.1% Tween. Fluorochrome-conjugated secondary antibodies (1:500 dilution) were added and incubated for 1 h at RT. After washing slides were washed with PBS and mounted using DAPI mounting solution (ThermoFisher, cat no. P36931). Slides were imaged using a Zeiss LSM 800 confocal laser scanning microscope.
Transmission Electron Microscopy
Carbon-coated grids (Electron Microscopy Sciences, EMS) was rendered hydrophilic by exposure to a 25 mA glow discharge for 20 s. For each AAV vector pre, 5 μl was adsorbed onto a grid for 1 minute, and stained with 1% uranyl acetate (EMS #22400) for 20s. Grids were examined in a TecnaiG2 Spirit BioTWIN and imaged with an AMT 2k CCD camera. Work was carried out at the Harvard Medical School Electron Microscopy Facility. Counts were performed as follows: 5 representative images of each vector prep were taken; all full and empty capsids were counted using the Count tool in Photoshop (CS6). Empty capsid percentage was calculated for each image, and plotted.
First, we constructed an AAV library plasmid which consisted of an AAV2 ITR-flanked expression cassette comprised of a chicken beta actin (CBA)-driven Cre recombinase and a p41promoter-driven AAV9 capsid (schematic in
To test the iTransduce library strategy, we performed a proof-of-concept selection to attempt to isolate AAV capsid variants with the ability to transduce brain cells after systemic injection. 1.27×1011 vector genomes (vg, 5×1012 vg/kg) of the library were injected intravenously through the tail vein of one adult male and one female Ai9 mouse and three weeks post-injection the mice were killed; a section of liver, brain, spleen, and kidney were cut and immunostaining of tdTomato was performed. We readily detected tdTomato positive cells (likely hepatocytes) in the liver, with scattered tdTomato positive cells (both neurons and astrocytes) in the brain as well as the other organs (
For the second round of selection, two Ai9 mice (one male, one female) were injected with the library rescued from round 1 (dose of 1.91×1010 vg, 7.64×1011 vg/kg) and sacrificed after three weeks. Prior to injection, sequence containing the variant region was amplified and sequenced by NGS to ensure a pre-existing bias had not been introduced into the vector pool (
To test whether these peptides expressed on the capsid of AAV could mediate efficient transgene expression by an AAV vector, we produced these capsids (AAV-S and AAV-F), packaging a single-stranded CBA promoter-driven GFP expression cassette (
Adult male C57BL/6J mice were injected via the lateral tail vein with a low dose or a high dose (1×1011 vg and 8×1011 vg of vector, respectively; approximately 4×1012 and 3.2×1013 vg/kg) of one of the following vectors: AAV9, AAV9-PHP.B, AAV-S and AAV-F (n=3 each). Three weeks post injection, mice were killed and organs harvested for endogenous (unstained) GFP fluorescence analysis. We quantitated the percent coverage of GFP signal in serial sagittal brain sections (3 sections were analyzed per animal). Remarkably, AAV-F demonstrated a 119-fold (p<0.0001) and 68-fold (p=0.0004) increased GFP fluorescence coverage compared to the parental AAV9 vector at 1×1011 vg and 8×1011 vg, respectively (
In order to get a detailed appreciation of the cell types being targeted by AAV-S and AAV-F, we next performed a series of co-immunostaining with GFP and markers of neurons (NeuN), astrocytes (Glutamine Synthetase, GS), microglia (Iba-1) and oligodendrocytes (Olig2). AAV-F and AAV-S, similar to the other two reference vectors, mainly transduced neurons and astrocytes (none of the variants appeared to effectively transduce microglial or oligodendroglial cells,
To better understand whether the high levels of GFP transgene expression in the brain with AAV-F corresponded to higher levels of AAV genomes in the brain, we isolated vector and murine genomic DNA from liver and brain and performed a qPCR on the 8×1011 vg dosed mice. AAV-F displayed a 20-fold enhancement (p<0.0001) in AAV genomes in the brain compared to AAV9 (
To investigate transgene expression in peripheral organs after systemic injection, we analyzed GFP fluorescence in the liver, heart, skeletal muscle, and retina (
Since transduction by AAV vectors in mice can vary substantially between sex and mouse strain, we examined whether the most efficient capsid, AAV-F could efficiently transduce brain after systemic injection of female C57BL/6 as well as male BALB/c. Mice were injected with 1×1011 vg (4×1012 vg/kg), and we observed robust and efficient transduction independent of strain or sex (
While iodixanol density gradient purification removes the majority of empty capsids, to examine if an excess of empty capsids in AAV-F could explain its increased biodistribution to the brain compared to AAV9, we performed transmission electron microscopy (TEM) on two separate preparations of AAV-F, and compared them to two independent preparations of AAV9. As seen in
Next, we tested whether AAV-F would have utility as a vector for CNS transduction via other routes of administration. We first tested AAV-S and AAV-F to mediate transgene expression in the brain after direct hippocampal injection of adult C57BL/6 mice. We found that both capsids achieve a widespread expression of GFP after direct injection, primarily in neurons (
Since the selection was performed in mice, we analyzed if the robust transduction characteristics of AAV-F also translated to human cells. Primary human stem cell-derived neurons were transduced with equal doses of AAV9, AAV-S and AAV-F, all encoding GFP. One week later they were fixed, stained with Class III β Tubulin and analyzed for the percentage of GFP positive neurons. Remarkably, AAV-F transduced 62% of neurons, 3-fold higher than AAV9 (p<0.05) (
Recent years have seen the development of new AAV vectors, designed to target specific organs and cell types with high efficacy. Many of these utilize 7-mer peptide insertions that modify the transduction properties of a particular AAV serotype. However, these vectors can often be repurposed to transduce other tissues, including those that are difficult to treat, such as the inner ear. Transducing certain cell types in the inner ear—such as hair cells—remains a challenge, with many conventional AAV vectors failing to consistently target one class of hair cells, outer hair cells (OHCs).
As noted above, one (AAV-F) showed great promise and high CNS expression after systemic injection, whereas the other (AAV-S) did not. While systemically injected AAV-S did not cross the blood-brain barrier effectively, it transduced a variety of tissues including heart, liver, and muscle. After direct injection into the brain, AAV-S displayed high local transduction efficiency in neurons, even at relatively low doses. To assay its transduction properties in the inner ear, we produced AAV-S encoding a single-stranded expression cassette driving EGFP under the CBA promoter, and injected it in neonatal (P1) mice via the round window. We found that AAV-S transduces hair cells of the cochlea extremely well. Both inner and outer hair cells were transduced with efficiencies of up to 100% and 99% (
2-3 rounds of selection for fibrocyte-targeting AAV capsids are performed in non-human primates, e.g., cynomolgus monkeys. To identify fibrocyte-selective, transduction-competent AAV capsid selection is performed by co-injecting the iTransduce AAV library along with AAV9-PHP.B which encodes a GJB2-floxed-STOP-tdTomato cassette (size fits inside AAV capsid). In NHP inner ear, AAV9-PHP.B-CBA-GFP transduces many cells of the cochlea including fibrocytes, HCs, and spiral ganglion neuron region. In this selection strategy (see
Alternatively or in addition, 2-3 rounds of selection for spinal cord cell targeting AAV capsids are performed in non-human primates, e.g., cynomolgus monkeys. To identify spinal cord-selective, transduction-competent AAV capsids, selection is performed by co-injecting the iTransduce AAV library along with AAV9 which encodes a CBA-floxed-STOP-mPlum cassette (size fits inside AAV capsid). In NHP spinal cord, AAV9-transduces cells including neurons and astrocytes. In this selection strategy (see
Once candidate capsids clones are identified, the capsids are vectorized as before, encoding a GFP cassette. Next, the capsids are tested for transduction of target cells after direct round window membrane (RMW) injection (e.g., as shown in 16A), or intrathecal injection (e.g., as shown in 16B).
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 62/825,703, filed on Mar. 28, 2019. The entire contents of the foregoing are incorporated herein by reference.
This invention was made with Government support under Grant Nos. AG047336 and DC017117 awarded by the National Institutes of Health. The Government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind |
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PCT/US2020/025720 | 3/30/2020 | WO | 00 |
Number | Date | Country | |
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62825703 | Mar 2019 | US |