Patent Application
20230193315
[0001.2] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 21, 2022, is named 60255-701-831-SL.txt and is 108,976 bytes in size.
This disclosure relates to viral vectors used in gene delivery. More specifically, this disclosure relates to a method for transcription-dependent directed evolution and adeno-associated virus (“AAV”) vectors that are selected by using this method.
Recombinant adeno-associated virus (“AAV”) vectors are among the most promising for in vivo gene delivery. The usefulness of AAV vectors has been expanded since a number of naturally occurring new serotypes and subtypes were isolated from human and non-human primate tissues. Gao et al., J Virol 78, 6381-6388 (2004) and Gao et al., Proc Natl Acad Sci USA 99, 11854-11859 (2002). Among the newly-identified AAV isolates, AAV serotype 8 (AAV8) and AAV serotype 9 (AAV9) have gained attention because AAV vectors derived from these two serotypes can transduce a variety of organs including the liver, heart, skeletal muscles and central nervous system with high efficiency following systemic administration. Ghosh et al., Mol Ther 15, 750-755 (2007); Pacak et al., Circ Res 99, 3-9 (2006); Inagaki et al., Mol Ther 14, 45-53 (2006); Zhu et al., Circulation 112, 2650-2659 (2005); Wang et al., Nat Biotechnol 23, 321-328 (2005); Nakai et al., J Virol 79, 214-224 (2005); and Foust et al., Nature Biotechnol 23, 321-328 (2009). This robust transduction by AAV8 and 9 vectors has been ascribed to strong tropism for these cell types, efficient cellular uptake of vectors, and/or rapid uncoating of virion shells in cells. Thomas et al., J Virol 78, 3110-3122 (2004). In addition, emergence of capsid-engineered AAV vectors with better performance has significantly broadened the utility of AAV as a vector toolkit. Asokan et al., Mol Ther 20, 699-708 (2012).
A proof-of-concept using AAV-mediated gene therapy has been shown in many preclinical animal models of human diseases. Phase I/II clinical studies have shown promising results for the treatment for hemophilia B (Nathwani et al., N Engl J Med 71, 1994-2004 (2014)), lipoprotein lipase deficiency (Carpentier et al., J Clin Endocrinol Metab 97, 1635-1644 (2012)), Leber congenital amaurosis (Jacobson et al., Arch Ophthalmol 130, 9-24 (2012) and Pierce and Bennett, Cold Spring Harb Perspect Med 5, a017285 (2015)), among others (reviewed in Mingozzi and High, Nat Rev Genet 12, 341-355 (2011) and Wang et al., Nat Rev Drug Discov 18, 358-378 (2019)).
Despite this promise, human studies have also revealed unexpected issues and potential challenges in AAV-mediated gene therapy. Manno et al., Nat Med 12, 342-347 (2006). In addition, despite rapid progress in our understanding of AAV biology and capsid-phenotype relationships (Adachi et al., Nat Commun 5, 3075, (2014); Grimm et al., Hum Gene Ther 28, 1075-1086, (2017); and Ogden et al., Science 366, 1139-1143, (2019)), there remain many desirable properties for clinical AAV vectors that we cannot rationally design.
To this end, high throughput screening methods for identifying novel AAV capsids with such desirable phenotypes have been employed. In particular, the development of in vivo AAV library selection strategies have produced a variety of designer AAV variants capable of highly efficient transduction of previously refractory cell types (reviewed in Kotterman and Schaffer, Nat Rev Genet 15, 445-451 (2014) and Grimm et al., Mol Ther 23, 1819-1831 (2015)).
The earliest attempts at in vivo library selection (1st Generation) relied on recovery of vector genome DNA from dissected tissue. Theoretically, this strategy results in recovery of both effective AAV variants, as well as AAV variants that mediate some, but not all of the steps required for vector-mediated transgene expression (
We therefore sought to develop a next-generation selection strategy (3rd Generation) with similar or better selective stringency as that provided by Cre-dependent selection, but without the need for Cre recombinase. In order to accomplish this goal, we developed the TRAnscription-dependent Directed Evolution system, or TRADE. In the transcription-dependent selection, we express the AAV cap gene as a non-coding antisense mRNA driven by a cell type-specific enhancer-promoter. Recovery of this antisense transcript by RT-PCR allows for stringent recovery of AAV cap genes at the level of vector-mediated mRNA expression in a specific cell type without the use of Cre recombinase. Targeting of different cell types merely requires cloning of a different cell type-specific enhancer-promoter into the plasmid construct. Thus, TRADE is a highly flexible system that can be applied in a wide variety of contexts, including the non-human primate context for development of enhanced AAV vectors for clinical gene therapy. Note that the same principle can be used for expressing AAV cap gene in an sense orientation. However, the sense strand approach results in expression of immunogenic capsid proteins in target cells and is therefore less ideal than the antisense strand approach employed by the TRADE system.
This disclosure provides a next-generation directed evolution strategy, termed TRAnscription-dependent Directed Evolution (“TRADE”), that selects for AAV capsid transduction at the level of cell type-specific or ubiquitous mRNA expression. The method described herein provides the following advantages over Cre recombination-based AAV targeted evolution (“CREATE”), the most contemporary methods for AAV capsid directed evolution reported in the literature. Deverman et al., Nat Biotech 34, 204-209 (2016). First, the CREATE system requires Cre expression, which can be attained either by exogenously-delivered Cre expression or by the use of Cre-transgenic animals. In contrast, the TRADE system does not require Cre-transgenic animals; therefore, it can be applied to animals and cultured cells derived from any animal species and can be readily adapted to large animals, including non-human primates. Second, unlike the CREATE system, in which the cell-type specific selection is applied at the level of AAV viral genome conversion from single-stranded DNA to double-stranded DNA, TRADE allows for cell type-specific selection at the level of AAV genome transcription. Therefore, the TRADE system can provide greater selective pressure than the CREATE system. Third, multiple directed evolution schemes (e.g., neuron-specific, astrocyte-specific, oligodendrocyte-specific, and microglia-specific) can be integrated into one AAV capsid library and selection for AAV vectors targeting each cell type can be performed in a single animal. Fourth, any cell type-specific or tissue/organ-specific enhancers/promoters or ubiquitous enhancers/promoters can be readily used for AAV capsid directed evolution aimed at identification of cell type-specific or ubiquitous novel AAV capsids with enhanced potency. Fifth, the TRADE methodology is not limited to the genus Dependoparvovirus, including the common AAVs that have been used for gene delivery, but can also be applied more broadly to the family Parvoviridae, including in the genera Bocaparvoviruses and Erythroparvoviruses other than AAV (e.g., bocaviruses), and even more broadly to an DNA virus.
This disclosure also provides novel AAV capsid mutants. TRADE technology was used to identify novel AAV vectors that mediate neuronal transduction in the brain following intravenous administration. Application of TRADE in C57BL/6J mice and a rhesus macaque resulted in the identification of new AAV capsids that can transduce neurons more efficiently and more specifically than AAV9 in the mouse and non-human primate brain following intravenous administration. In addition, we identified a novel AAV capsid that can transduce an undefined cell population or populations, that reside in the lung and are potentially of neuronal origin, 5 to 18 times better than the AAV9.
The present disclosure also provides a method to prevent splicing of antisense mRNA of the AAV capsid gene. Antisense pre-mRNA transcribed from the AAV cap gene open reading frame (“ORF”) can be spliced making (a) truncated mRNA species. To our knowledge, this is a new discovery that has never previously been reported. Such splicing has the potential to hinder effective recovery of full-length antisense mRNA of the AAV cap ORF, which is essential for TRADE when a wide region of the cap ORF is mutagenized. This disclosure provides a novel strategy to prevent splicing of antisense mRNA of the cap gene.
The TRADE system described herein uses antisense mRNA to recover capsid sequence information, TRADE using sense strand mRNA (i.e., sense strand TRADE) is also feasible using the same principle. However, it should be noted that the sense strand TRADE approach results in expression of immunogenic capsid proteins in target cells and therefore is presumably less ideal than the antisense strand approach.
In some embodiments, the present disclosure provides a TRADE system that allows directed evolution of the AAV capsid using antisense mRNA of the cap ORF expressed in a cell type-specific or ubiquitous manner. Such a system does not require Cre-transgenic animals. Therefore, it can be applied to cell type-specific AAV capsid evolution in large animals, including non-human primates, for which Cre-transgenic strains are not readily available. Any cell type-specific or tissue/organ-specific enhancers/promoters or ubiquitous enhancers/promoters can be readily applied to the system with no requirement of transgenesis. The cell type-specific selection is given at the mRNA level. In certain embodiments, multiple directed evolution schemes may be combined into one directed evolution scheme. For example, selection of neuron-specific AAV capsids, astrocyte-specific AAV capsids, oligodendrocyte-specific AAV capsids and microglia-specific AAV capsids based on cell type-specific transgene mRNA expression can be performed simultaneously in a single animal.
In some embodiments, the present disclosure provides a sense strand TRADE system that allows directed evolution of the AAV capsid using mRNA of the cap ORF expressed in a cell type-specific or ubiquitous manner that is capable of expressing AAV capsid proteins in target cells. The sense strand TRADE has the same advantages of those antisense strand TRADE presented with data here in that it does not require Cre-transgenic animals, cell type-specific selection is given at the mRNA level, and it is capable of combining multiple directed evolution schemes into one directed evolution round done in a single animal. However, the possible disadvantage is that immunogenic AAV capsid proteins may be unavoidably expressed persistently in target cells, which may result in undesired consequences in the capsid selection process.
In some embodiments, the present disclosure also provides novel AAV capsids. In certain embodiments, these novel AAV capsids can transduce brain neurons several times better than AAV9 in C57BL/6J mice following intravenous injection. In certain embodiments, the novel AAV capsids transduced up to 8 times better than AAV9 in C57BL/6J mice following intravenous injection. The neuronal transduction levels may be greatly enhanced compared to AAV9 although they may not attain the levels obtained with AAV PHP.B. In certain embodiments, the novel AAV capsids may transduce brain neurons more efficiently than AAV PHP.B.
In some embodiments, this disclosure provides novel AAV capsids that can transduce brain neurons several times better than AAV9 following intravenous injection in BALB/cJ mice. In certain embodiments, the novel AAV capsids can transduce brain neurons up to 7 times better than AAV9 following intravenous injection in BALB/cJ mice. The transduction levels are much higher than AAV PHP.B.
In some embodiments, this disclosure provides novel AAV capsids that can transduce brain neurons several times better than AAV9 in rhesus macaques following intravenous injection. In certain embodiments, the novel AAV capsids can transduce brain neurons up to 4 times better than AAV9 in rhesus macaques following intravenous injection. These transduction levels are better than AAV PHP.B.
In some embodiments, the disclosure provides AAV capsids that can transduce the pulmonary cells with neuronal cell marker expression several times better than AAV9. In certain embodiments, the AAV capsids can transduce such cells up to 17 times better than AAV9.
In some embodiments, the novel AAV capsids exhibit a liver-detargeting phenotype.
In some embodiments, the disclosure provides codon-modified AAV cap sequences that are not spliced when expressed in an antisense direction. We have observed that unmodified AAV cap ORFs are spliced when expressed in an antisense direction (e.g., AAV1, AAV3 and AAV9). In contrast, some of the codon-modified AAV cap ORFs described in this disclosure are not spliced. Based on the knowledge we have developed about the putative splice donor and acceptor sites, it has become possible to design such non-spliced versions of AAV cap ORFs. The use of such non-spliced cap ORFs may be used for directed evolution using the TRADE system when mutagenesis of the cap gene takes place over a wide region of the cap ORF.
The term “AAV vector” as used herein means any vector that comprises or derives from components of AAV and is suitable to infect mammalian cells, including human cells, of any of a number of tissue types, such as brain, heart, lung, skeletal muscle, liver, kidney, spleen, or pancreas, whether in vitro or in vivo. The term “AAV vector” may be used to refer to an AAV type viral particle (or virion) comprising at least a nucleic acid molecule encoding a protein of interest.
Additionally, the AAVs disclosed herein may be derived from various serotypes, including combinations of serotypes (e.g., “pseudotyped” AAV) or from various genomes (e.g., single-stranded or self-complementary). In particular embodiments, the AAV vectors disclosed herein may comprise desired proteins or protein variants. A “variant” as used herein refers to an amino acid sequence that is altered by one or more amino acids. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. Alternatively, a variant may have “nonconservative” changes, e.g., replacement of a glycine with a tryptophan. Analogous minor variations may also include amino acid deletions or insertions, or both.
Nucleotide sequences, such as polynucleotides, encoding proteins of the present disclosure are provided herein. The nucleotides of the present disclosure can be composed of either RNA or DNA. The disclosure also encompasses those polynucleotides that are complementary in sequence to the polynucleotides disclosed herein.
Because of the degeneracy of the genetic code, a variety of different polynucleotide sequences can encode the proteins of the present disclosure. In addition, it is well within the skill of a person trained in the art to create alternative polynucleotide sequences encoding the same, or essentially the same, proteins disclosed herein. These variant or alternative polynucleotide sequences are within the scope of the current disclosure. As used herein, references to “essentially the same sequence” refers to one or more sequences that encode amino acid substitutions, deletions, additions, or insertions that do not eliminate the detectability of the polypeptide encoded by the polynucleotides of the present disclosure.
The current disclosure also includes variants of the polynucleotides and polypeptides disclosed herein. Variant sequences include those sequences wherein one or more peptides or nucleotides of the sequence have been substituted, deleted, and/or inserted.
Polynucleotide and polypeptide sequences of the current disclosure can also be defined in terms of particular identity and/or similarity with certain polynucleotides and polypeptides described herein. The sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%. The identity and/or similarity of a sequence can be 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical as compared to a sequence disclosed herein. Unless otherwise specified, as used herein percent sequence identity and/or similarity of two sequences can be determined using the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990). BLAST searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used.
Methods of producing AAV vectors as disclosed herein are well known in the art, including methods, for example, of using packaging cells, auxiliary viruses or plasmids, and/or baculovirus systems. See, e.g., Samulski et al., J. Virology 63, 3822 (1989); Xiao et al., J. Virology 72, 2224 (1998); Inoue et al., J. Virology 72, 7024 (1998); WO1998/022607; and WO2005/072364.
Methods of producing pseudotyped AAV vectors are also known (see, e.g., WO00/28004), as well as various modifications or formulations of AAV vectors, to reduce their immunogenicity upon in vivo administration (see, e.g., WO01/23001; WO00/73316; WO04/112727; WO05/005610; and WO99/06562). In some embodiments, AAV vectors may be prepared or derived from various serotypes of AAVs which may be mixed together or mixed with other types of viruses to produce chimeric (e.g., pseudotyped) AAV viruses.
In particular embodiments, the AAV vector may be a human serotype AAV vector. In such embodiments, a human AAV may be derived from any known serotype, e.g., from any one of serotypes 1-11, for instance from AAV1, AAV2, AAV4, AAV6, or AAV9.
The AAV vectors disclosed herein may include a nucleic acid encoding a protein of interest. In various embodiments, the nucleic acid also may include one or more regulatory sequences allowing expression and, in some embodiments, secretion of the protein of interest, such as e.g., a promoter, enhancer, polyadenylation signal, an internal ribosome entry site (“IRES”), a sequence encoding a protein transduction domain (“PTD”), a 2A peptide, and the like. Thus, in some embodiments, the nucleic acid may comprise a promoter region operably linked to the coding sequence to cause or improve expression of the protein of interest in infected cells. Such a promoter may be ubiquitous, cell- or tissue-specific, strong, weak, regulated, chimeric, etc., for example, to allow efficient and stable production of the protein in the infected tissue. The promoter may be homologous to the encoded protein, or heterologous, although generally promoters of use in the disclosed methods are functional in human cells. Examples of regulated promoters include, without limitation, Tet on/off element-containing promoters, rapamycin-inducible promoters, tamoxifen-inducible promoters, and metallothionein promoters. Other promoters that may be used include promoters that are tissue specific for tissues such as kidney, spleen, and pancreas. Examples of ubiquitous promoters include viral promoters, particularly the CMV promoter, the RSV promoter, the SV40 promoter, etc., and cellular promoters such as the phosphoglycerate kinase (PGK) promoter and the β-actin promoter.
In some embodiments of the AAV vectors disclosed herein, one or more feedback elements may be used to dampen over-expression of the protein of interest. For example, some embodiments of the AAV vectors may include one or more siRNA sequences that would target the exogenous transcript. In other embodiments, the AAV vector may include one or more additional promoters that may be recognized by inhibitory transcription factors. In various embodiments, the AAV vectors disclosed herein may comprise a construct that may create a homoeostatic feedback loop that may maintain expression levels of the protein of interest at a physiological level.
In some embodiments of the AAV vectors disclosed herein, genome editing machinery may be used to genetically modify cellular genome DNA or mRNA transcripts at a site-specific manner. Komor et al., Cell 168, 20-36 (2017); and Katrekar et al., Nature Methods 16:239-242, 2019. For example, some embodiments of the AAV vectors may include a CRISPR-associated enzyme such as Cas9, a DNA base editor, an RNA editase and/or guide RNA (gRNA) to modify nucleic acid in cells in a site-specific manner. In addition, AAV vectors may contain a homology repair template (HDR) for genome editing.
In various embodiments, the AAV vectors disclosed herein can comprise a nucleic acid that may include a leader sequence allowing secretion of the encoded protein. In some embodiments, fusion of the transgene of interest with a sequence encoding a secretion signal peptide (usually located at the N-terminal of secreted polypeptides) may allow the production of the therapeutic protein in a form that can be secreted from the transduced cell. Examples of such signal peptides include the albumin, the β-glucuronidase, the alkaline protease or the fibronectin secretory signal peptides.
As described herein, effective and long-term expression of therapeutic proteins of interest in brain, heart, lung, skeletal muscle, kidney, spleen, or pancreas can be achieved with non-invasive techniques, through peripheral administration of certain AAV vectors, such as a non-AAV9 vector with AAV9 sequences. Such peripheral administration may include any administration route that does not necessitate direct injection into brain, heart, lung, skeletal muscle, kidney, spleen, or pancreas. More particularly, peripheral administration may include systemic injections, such as intramuscular, intravascular (such as intravenous,) intraperitoneal, intra-arterial, or subcutaneous injections. In some embodiments, peripheral administration also may include oral administration (see, e.g., WO96/40954), delivery using implants, (see, e.g., WO01/91803), or administration by instillation through the respiratory system, e.g., using sprays, aerosols or any other appropriate formulations.
In various embodiments, the desired doses of the AAV vectors may be adapted by the skilled artisan, e.g., depending on the disease condition, the subject, the treatment schedule, etc. In some embodiments, from 105 to 1012 viral genomes are administered per dose, for example, from 106 to 1011, from 107 to 1011, or from 108 to 1011. In other embodiments, exemplary doses for achieving therapeutic effects may include virus titers of at least about 105, 106, 107, 108, 109, 1010 or 1011 viral genomes or more. Virus titer may also be expressed in terms of transducing units, which may be readily calculated by those of skill in the art.
In various embodiments, the AAV vectors disclosed herein may be administered in any suitable form, for instance, either as a liquid solution or suspension, as a solid form suitable for solution or suspension in liquid prior to injection, as a gel or as an emulsion. The vectors may be formulated with any appropriate and pharmaceutically acceptable excipient, carrier, adjuvant, diluent, etc. For instance, for injection, a suitable carrier or diluent may be an isotonic solution, a buffer, sterile and pyrogen-free water, or, for instance, a sterile and pyrogen-free phosphate-buffered saline solution. For inhalation, the carrier may be in particulate form.
The vectors may be administered in a “therapeutically-effective” amount, e.g., an amount that is sufficient to alleviate (e.g., decrease, reduce) at least one of the symptoms associated with a disease state, or to provide improvement in the condition of the subject. In some embodiments, repeated administrations may be performed, for instance using the same or a different peripheral administration route and/or the same vector or a distinct vector.
The following examples are for illustration only. In light of this disclosure, those of skill in the art will recognize that variations of these examples and other embodiments of the disclosed subject matter are enabled without undue experimentation.
We applied the TRADE system in both C57BL/6J mice and a rhesus macaque in order to identify novel AAV capsids that efficiently transduce brain neurons following systemic delivery. The TRADE system utilizes a plasmid construct containing an overlapping bicistronic AAV genome flanked by ITR sequences (
We first tested the ability of the TRADE system to recover the sequence of the AAV cap gene from cell type-specific antisense mRNA using an AAV-PHP.B-hSynl-GFP-TRADE vector (
With the successful establishment of the TRADE system, we performed two AAV capsid directed evolution experiments; one used 8-week-old male C57BL/6J mice and the other used one 8-month-old male rhesus macaque. We produced an AAV9-N272A-hSynl-GFP-TRADE-Lib library composed of AAV9-derived mutant capsids that have a GGGS(N8)GGGGS (SEQ ID NO:2) peptide insertion at the position of Q588 where N8 represents a random 8-mer peptide encoded by (NNK)8. For the peptide insertion, Q588 was substituted with each peptide sequence. The diversity of the AAV library was at least 107. In the mouse directed evolution experiment, we infused the AAV library via the tail vein at a dose of 3 x 1011 vector genomes (vg) per mouse. For the second round of selection, we injected the AAV library at a dose of 1×1012, 1×1011, 1×1010, or 1x109 vector genomes (vg) using two mice. For the third round of selection, we injected the AAV library at a dose of 1×1011 vg using two mice. We harvested brain tissues twelve days after injection, and separated them into three regions, i.e., the cerebrum, the cerebellum and the brain stem. Only the cerebrum samples were used for the directed evolution experiments. We extracted total RNA from the cerebrum, reverse-transcribed the RNA using an oligo dT primer, and amplified the peptide region including the flanking regions by a pair of the cap gene-specific PCR primers. The RT-PCR products were then used to create the next AAV9-N272A-hSynl-TRADE-Lib plasmid library, which was subsequently used to produce the next AAV9-N272A-hSynl-TRADE-Lib virus library. For the second and third round selection, we packaged an AAV9-N272A-hSynl-TRADE-Lib genome that was devoid of the GFP ORF. In the non-human primate directed evolution experiment, we infused the AAV9-N272A-hSynl-GFP-TRADE-Lib library via the saphenous vein at a dose of 2.0 x 1012 vg per kg. Twelve days post-injection, the whole brain was harvested and sliced using a brain matrix, treated with RNAlater (Thermo Fisher Scientific), and stored frozen. Total RNA was then extracted from the following brain regions: frontal cortex, occipital cortex, cerebellum (Purkinje and granular layers), medulla, pons, frontal cortex, hypothalamus, thalamus, cingulate gyrus, caudate nucleus, putamen, hippocampus, and preoptic area. We retrieved the peptide sequences by RT-PCR in the same manner as described above except that we performed nested PCR to obtain PCR products sufficient for the downstream Illumina and Sanger sequencing procedures. For some samples, we cloned the first PCR products directly into a plasmid backbone without performing nested PCR for Sanger sequencing. Following three rounds of selection in mice (Table 1) and one round of selection in non-human primate, we identified a number of potentially transduction-enhancing peptides inserted into the AAV9 capsids (Table 2). We then generated a barcoded AAV library and utilized DNA/RNA Barcode-Seq technology, previously developed in the Nakai lab (Adachi et al. Nat Commun 5, 3075 (2014); and PCT/US2017/068050), to compare the transduction efficiency, tropism/biodistribution, and pharmacokinetics of 26 selected novel AAV variants (Table 3) following intravenous administration in two commonly used mouse lines (C57BL/6J and BALB/cJ) and one rhesus macaque. As a result, we have found: (1) Some of the novel variants identified by TRADE technology, in particular AAV9-N272A-TTNLAKNS (HN1) and AAV9-N272A-QQNGTRPS (HN2), performed up to 8 times better than AAV9 in the brain of C57BL/6J mice (
In the course of the experiment, when the AAV9 cap gene ORF was expressed in an antisense orientation in HEK293 cells or Neuro2a cells, the majority of the antisense AAV9 cap gene mRNA-derived RT-PCR products were truncated by approximately 1.7 kb (
Potential splicing of the cap ORF-derived antisense mRNA is scientifically intriguing, but may hinder the TRADE system when the full-length cap ORF sequence needs to be recovered from antisense mRNA. To overcome this potential issue, we introduced silent mutations that presumably disrupt the conserved sequences at exon-intron junctions and branching points. To demonstrate proof of principle of this approach, we introduced silent mutations into the AAV9 cap ORF contained in the plasmid, pAAV9-N272A-PHP.B-hSyn1-GFP-TRADE, that disrupt the splice acceptor (SA) consensus sequence (pAAV9NS1 construct), the splice donor (SD) consensus sequence (pAAV9NS2 construct), and both the splice acceptor and donor consensus sequences (pAAV9NS3 construct). Please note that NS stands for “non-spliced.” The method we use to disrupt these consensus sequences is described below.
We codon-optimize the AAV cap ORF sequence for human cell expression.
To identify potential splice donor and acceptor sites on antisense mRNA derived from the cap ORFs, we develop and use our proprietary database of potential splice donor and acceptor sites on antisense mRNA based on our experimental and bioinformatics observations (i.e.,
We destroy the GT (splice donor) and / or AG (splice acceptor) consensus sequence by changing at least one nucleotide using the codon-optimized sequence. If the codon-optimized sequence is not applicable, we use an alternative nucleotide(s) that can destroy the consensus sequence.
We remove a stretch of T’s upstream of the splice acceptor sites by introducing silence mutations based on the codon-optimized sequence. If the codon-optimized sequence is not sufficient to destroy a stretch of T’s, we use alternative nucleotides.
We also avoid G at the exon termini as much as possible.
Using several programs that can predict branching points (e.g., Human Splicing Finder (Desmet, Hamroun et al. 2009)), we identify potential branching points and replace them with the codon-optimized sequence. If the degree of nucleotide changes attainable by this method is not sufficient, we introduce alternative nucleotides to disrupt potential branching points.
With this method, we have created AAV9NS1 (SA, destroyed), AAV9NS2 (SD, destroyed) and AAV9NS3 (both SD and SA, destroyed) cap ORFs (
The TRADE method described herein uses antisense mRNA for viral protein evolution to establish the proof-of-principle and to show successful reduction of the method to practice. The TRADE system can also utilize mRNA in a sense orientation as long as the viruses can be produced and potential expression of viral proteins in target cells during the directed evolution procedure does not hinder successful evolution of novel capsids.
Additional information related to nucleic acid splicing and AAV may be found in Desmet et al., Nucleic Acids Res 37, e67 (2009); Matsuzaki et al., Neurosci Lett 665, 182-188 (2018); and Hordeaux et al., Mol Ther26, 664-668 (2018).
All references cited in this disclosure are incorporated by reference in their entirety.
[0001.1] This application is the U.S. National Phase application of PCT/US2020/016273, filed on Jan. 31, 2020, which claims the benefit of U.S. Provisional Application No. 62/799,603 filed on Jan. 31, 2019, all of which are incorporated herein by reference in their entirety.
This invention was made with government support under Grant No. NS088399 awarded by the National Institutes of Health/National Institute of Neurological Disorders and Stroke. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/016273 | 1/31/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62799603 | Jan 2019 | US |
