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WO2022120103A1 - Ingénierie d'aav orthogonal immunitaire et de furtif immunitaire crispr-cas - Google Patents

Ingénierie d'aav orthogonal immunitaire et de furtif immunitaire crispr-cas Download PDF

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WO2022120103A1
WO2022120103A1 PCT/US2021/061682 US2021061682W WO2022120103A1 WO 2022120103 A1 WO2022120103 A1 WO 2022120103A1 US 2021061682 W US2021061682 W US 2021061682W WO 2022120103 A1 WO2022120103 A1 WO 2022120103A1
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protein
sequence
cas9
mutations
virus
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Prashant MALI
Nathan Palmer
Aditya Kumar
Amanda SUHARDJO
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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Priority to JP2023532295A priority Critical patent/JP2023551678A/ja
Priority to EP21901490.9A priority patent/EP4256046A4/fr
Publication of WO2022120103A1 publication Critical patent/WO2022120103A1/fr
Priority to US17/851,972 priority patent/US20220364073A1/en
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1089—Design, preparation, screening or analysis of libraries using computer algorithms
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
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    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
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    • C40B40/04—Libraries containing only organic compounds
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
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    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Described herein are methods for engineering proteins and viruses to reduce their immunogenicity, proteins and viruses made by using said methods, including proteins having Cas9 like activity and viruses having AAV5 like activity.
  • Immunogenicity is a major concern for protein-based therapeutics, particularly those derived from non-human species. Induction of the immune response can render treatments ineffective and cause serious, even life-threatening side effects.
  • One strategy to overcome this issue is to mutate particularly immunogenic epitopes in the therapeutic target. However, this strategy is hindered by the ability of the adaptive immune system to recognize multiple epitopes across large regions of the antigen. While epitope deletion efforts to date have focused on a few major antibody binding sites, it is not possible to make these studies comprehensive due to the vast possible epitope space.
  • Variant library screening has proven to be an effective approach to protein engineering but applying it in this case faces several technical challenges .
  • One problem is the vast mutational space created by the need for full combinatorial libraries .
  • the disclosure provides a method that overcomes the library s i ze constraints in both the number of unique members and the length of the mutageni zed region .
  • the method comprises selecting target regions within a protein of interest us ing software which predicts HLA-binding and peptide immunogenicity .
  • To generalize immunogenicity predictions and select appropriate targets an approximation of global HLA allele frequencies is generated us ing data from the Allele Frequency Net Database . These frequencies were used to scale immunogenicity predictions such that the top hits are the peptides li kely to be the most immunogenic epitopes for the largest number of people globally .
  • the disclosure provides a method for engineering a protein or virus to be less immunoreactive, comprising: identifying target regions of the polynucleotide sequence encoding a protein or virus that are predicted to have human leukocyte antigen (HLA) -binding and/or peptide immunogenicity; identifying single nucleotide polymorphisms (SNPs) or mutations in the targeted region and other regions that are not deleterious to the functioning of the protein; screening a library assembled using standard synthesis and assembly methods by applying the above identifying criteria to find functional variants of the protein or virus; sequencing the functional variants of the protein or virus; mapping genotype to phenotype from the sequences of the functional variants to identify variant candidates that are likely functionally active and have mutations that result in the protein or virus exhibiting less immunogenicity.
  • HLA human leukocyte antigen
  • the protein is a CRISPR associated protein.
  • the CRISPR associated protein is a Cas9.
  • the Cas9 is Streptococcus pyogenes Cas9 (SpCas9) .
  • the target regions are identified using a model that predicts human leukocyte antigen (HLA) -binding and peptide immunogenicity.
  • the prediction model is selected from NetMHC, MHCAttnNet, MHCSeqNET, ACME, NetMHCpan EL 4.1, NetMHCstabpan, SMM, SMMPMBEC, PickPocket, Comblib_Sidney2008, NetMHCcons, MHCf lurry 2.0, and IConMHC.
  • the SNPs or mutations are identified by using phylogenetic methods to scan natural variation among naturally occurring SpCas9, mutations generated in the course of research and engineering efforts, and the Cas9 orthologs of closely related bacterial species.
  • the SNPS or mutations are identified, or further identified, by using immunological prediction of candidate mutations to ensure significant loss of immunogenicity within the targeted region in order to preserve function while reducing immunogenicity.
  • the virus is an adeno-associated virus (AAV) .
  • AAV adeno-associated virus
  • the AAV is selected from AAV1, AAV2, AAV5, AAV6, AAV7 , and AAV8.
  • the AAV is AAV5.
  • the target regions are identified by aligning conserved sequence regions across AAV serotypes.
  • the SNPs are identified by aligning the sequences of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more AAV variants that have been sequenced from natural or engineered sources.
  • the SNPs are located in the target regions.
  • long-read sequencing technologies capable of sequencing the entire sequence of each protein or viral variant in one sequencing reaction is used to sequence the functional variants of the protein or virus.
  • the long-read sequencing technologies is capable of generating 10-15 Gb of sequencing reads per run.
  • a method disclosed herein further comprises the step of evaluating the immunoreactivity of variant candidates in one or more immunoassays.
  • the one or more immunoassays comprise detecting the presence of antibodies to the variant candidates (AVA antibodies) , when the variant candidates are administered in vivo to an animal.
  • the one or more immunoassays comprise an enzyme-linked immunosorbent assays (ELISAs) , electrochemiluminescence (ECL) assays and/or antigen-binding tests, wherein the one or more immunoassays utilize AVA antibodies.
  • ELISAs enzyme-linked immunosorbent assays
  • ECL electrochemiluminescence
  • the disclosure provides an isolated polypeptide encoded by a polynucleotide sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, wherein the polypeptide has Cas9 like activity and is less immunogenic that SEQ ID NO: 2.
  • the polynucleotide encodes a polypeptide of SEQ ID NO: 2 having one or more mutations selected from the group consisting of: P28L, L237C, Y286Q, S318 (H/C) , S368C, F498T, L514 (T/G) , L616G, L623Q, L636D, F704A, L727 (P/G) , L816D, Y1016 (K/G) , L1245G, I1273Q, L1282 (A/E) , and Y1294Q.
  • the disclosure contemplates any combination of the foregoing mutations to SEQ ID NO: 2, wherein the number of mutations comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 mutations as recited above.
  • the disclosure contemplates any combination of the foregoing mutations to SEQ ID NO: 2, wherein the number of mutations comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, or 18 mutations as recited above.
  • the polypeptide may have 1-10 additional conservative mutations at positions other than those set forth above.
  • the disclosure also provides an isolated polypeptide having a sequence that has at least 90%, 95%, 97%, 98%, or 99% sequence identity to the sequence presented in SEQ ID NO: 2 wherein the protein has Cas9 like activity and is immuno-silenced compared to a polypeptide of SEQ ID NO: 2.
  • the polypeptide comprises the sequence of SEQ ID NO: 2 and having one or more mutations selected from the group consisting of: P28L, L237C, Y286Q, S318 (H/C) , S368C, F498T, L514 (T/G) , L616G, L623Q, L636D, F704A, L727 (P/G) , L816D, Y1016 (K/G) , L1245G, I1273Q, L1282 (A/E) , and Y1294Q.
  • the disclosure contemplates any combination of the foregoing mutations to SEQ ID NO: 2, wherein the number of mutations comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 mutations as recited above.
  • the disclosure contemplates any combination of the foregoing mutations to SEQ ID NO: 2, wherein the number of mutations comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, or 18 mutations as recited above.
  • the polypeptide may have 1-10 additional conservative mutations at positions other than those set forth above .
  • the disclosure also provides a virus comprising a viral capsid encoded by a polynucleotide having 90-99% sequence identity to SEQ ID NO: 49 and encoding a viral capsid polypeptide having AAV5 like activity and is immuno-silenced compared to a wild-type viral capsid of SEQ ID NO: 50.
  • the viral capsid comprises a sequence of SEQ ID NO: 50 and comprises two or more mutation selected from the group consisting of R42G, P47L, Y49C, G55S, N56H, G57S, D59Y, Y89H, L90 (P/I) , A95V, D96G, E98 (K/Q) , F99L, T107A, S108P, Q119 (R/E) , R123 (L/T) , V124A, V131A, E132 (G/R) , E133 (Q/D) , G134 (V/S) , T137A, A214V, S222T, T223 (A/K) , S267A, Y272H, F273L, W287R, L290P, I291V, I309V, K312 (E/R) , N400D, F402L, F413L, S415T, S416(M/G) , Q
  • the disclosure contemplates any combination of the foregoing mutations to SEQ ID NO: 50, wherein the number of mutations comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more,
  • the viral capsid comprises no more than 60 mutations.
  • the viral capsid may have 1-10 additional conservative mutations at positions other than those set forth above.
  • the disclosure also provides for an AAV system comprising the virus disclosed herein that has AAV5 like activity.
  • the AAV system is used for gene therapy.
  • Figure 1A-B provides (A) schematic of library design methodology; (B) tables showing the location, codon information, etc. for the generation of a library of hCas9 constructs using a method disclosed herein. As indicated in the lower table, there were 56 total constructs generated from a library having the size of 995328.
  • Figure 2 provides a library construction schematic.
  • Figure 3 provides Immunogenicity scores of Cas orthologs.
  • Figure 4 provides Cas9 functional screen schematic.
  • Figure 5 provides mutation density distribution of Cas9 library before and after screening.
  • Figure 6 provides HeLa cells transduced with Cas9 and HPRT1 or non-targeting guide were treated with 0-14 pg/mL 6TG. Viable cells are shown by crystal violet staining.
  • Figure 8 provides pre- and post-screen allele frequencies at each of the 18 mutation sites. Each site shows enrichment of the wild-type allele, but most sites retain a substantial fraction of mutant alleles.
  • Figure 9 provides neighbor score as a metric for differentiating true positive hits. Left: Neighbor score is positively correlated with screen score. Right: Divergence between neighbor score and screen score decreases as read coverage increases .
  • Figure 10 shows a network diagram of screen hits.
  • Nodes are Cas9 variant hits with high coverage in each replicate and enrichment greater than wild-type. Edges between nodes correspond to the distance between genotypes and are weighted as 1/n for n ⁇ 5 where n is the number of non-shared mutations.
  • Figure 11 shows HDR efficiencies for wildtype and variant Cas as quantified by FACS.
  • Figure 12 provides NHEJ efficiencies for wildtype and variant Cas as quantified by NGS.
  • Figure 13 provides Correlation between pLDDT and epistasis in our Cas9 screen (top left) , as well as third party double mutant datasets for GFP (top right, DOI:
  • Figure 14 shows maximum likelihood cladogram of AAVs including the main human serotypes and new tested orthologs.
  • Figure 15 provides Viral formation titers of AAV orthologs relative to AAV5.
  • Figure 16 provides tables showing the location, codon information, etc. for the generation of a library of AAV5 constructs using a method disclosed herein. As indicated in the lower table, there were 40 total constructs generated from a library having the size of 2097152.
  • Figure 17 provides a DNA sequence (SEQ ID NO: 49) listing an exemplary AAV5 construct that has modified so as to be less immunoreactive .
  • Figure 18 provides an AA sequence (SEQ ID NO: 50) of an exemplary AAV5 construct that has modified so as to be less immunoreactive .
  • the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value.
  • the term "about” meaning within an acceptable error range for the particular value can be assumed.
  • the ranges and/or subranges can include the endpoints of the ranges and/or subranges. In some cases, variations can include an amount or concentration of 20%, 10%, 5%, 1 %, 0.5%, or even 0.1 % of the specified amount.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • AAV adeno-associated virus
  • AAV adeno-associated virus
  • AAV refers to a member of the class of viruses associated with this name and belonging to the genus dependoparvovirus , family Parvoviridae . Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types.
  • Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 or 12 serotypes, e.g. , AAV2, AAV5, and AAV8, or variant serotypes such as AAV-DJ.
  • the AAV structural particle is composed of 60 protein molecules made up of VP1, VP2 and VP3. Each particle contains approximately 5 VP1 proteins, 5 VP2 proteins and 50 VP3 proteins ordered into an icosahedral structure.
  • Non-limiting exemplary VP1 sequences useful in the methods disclosed herein are provided below.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g. , hydroxyproline, y-carboxyglutamate , and O- phosphoserine .
  • an amino acid analog refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i . e . , a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g.
  • an amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • non-naturally occurring amino acid and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.
  • one or more D-amino acids can be used in various peptide compositions of the disclosure.
  • the disclosure provides various peptides that are useful for treating various diseases and infections. These peptides can comprise naturally occurring amino acid. In other embodiments, the peptides can comprise non-natural amino acids. The use of non-natural amino acids can improve the peptides stability, decrease degradation and/or improve biological activity. For example, in some embodiments, one or more D-amino acids. In other embodiments, retroinverso peptides are contemplated using various amino acid configurations.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Cas9 refers to a CRISPR-associated, RNA-guided endonuclease such as Streptococcus pyogenes Cas9 (spCas9; see Accession Number Q99ZW2.1, the sequence of which is incorporated herein by reference) and orthologs and biological equivalents thereof.
  • Biological equivalents of Cas9 include, but are not limited to, C2cl from Alicyclobacillus acideterrestris and Cpfl (which performs cutting/cleaving functions analogous to Cas9) from various bacterial species including Acidaminococcus spp. and Francisella novicida U112.
  • Cas9 may refer to an endonuclease that causes double stranded breaks in DNA, a nickase variant such as a RuvC or HNH mutant that causes a single stranded break in DNA, as well as other variations such as deadCas-9 (“dCas9”) , which lack endonuclease activity.
  • Cas9 may also refer to "split-Cas9" in which Cas9 is split into two halves - C-terminal Cas9 (C-Cas9) and an N- terminal Cas-9 (N-Cas9) - which can be fused with two intein moieties. See, e.g. , U.S. Pat. No. 9, 074, 199 Bl; Zetsche et al.
  • Non-limiting examples of commercially available sources of SpCas9 comprising plasmids can be found under the following AddGene reference numbers:
  • 48138 PX458; SpCas 9-2A-EGFP and single guide RNA
  • 62988 PX459; SpCas 9-2A-Puro and single guide RNA
  • 48873 PX460; SpCas9n (D10A nickase) and single guide RNA
  • 48140 PX461; SpCas 9n-2A-EGFP (D10A nickase) and single guide RNA;
  • CRISPR refers to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) .
  • CRISPR may also refer to a technique or system of sequence-specific genetic manipulation relying on the CRISPR pathway.
  • a CRISPR recombinant expression system can be programmed to cleave a target polynucleotide using a CRISPR endonuclease and a guideRNA.
  • a CRISPR system can be used to cause double stranded or single stranded breaks in a target polynucleotide.
  • a CRISPR system can also be used to recruit proteins or label a target polynucleotide.
  • CRISPR-mediated gene editing utilizes the pathways of nonhomologous end- joining (NHEJ) or homologous recombination to perform the edits.
  • NHEJ nonhomologous end- joining
  • homologous recombination to perform the edits.
  • domain can refer to a particular region of a larger molecule (e.g. , a particular region of a protein or polypeptide) , which can be associated with a particular function.
  • a domain which binds to a cognate can refer to the domain of a protein that binds one or more receptors or other protein moieties.
  • a corresponding coding sequence for a particular polypeptide domain can be referred to as a polynucleotide domain.
  • encode as it is applied to polynucleotides can refer to a polynucleotide which is said to "encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof.
  • antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • expression can refer to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.
  • the term "functional" may be used to modify any molecule, biological, or cellular material to intend that it accomplishes a particular, specified effect.
  • gRNA or "guide RNA” as used herein refers to the guide RNA sequences used to target specific genes for correction employing the CRISPR technique.
  • Techniques of designing gRNAs and donor therapeutic polynucleotides for target specificity are well known in the art. For example, Doench, J. , et al. Nature biotechnology 2014; 32 (12) : 1262-7, Mohr, S. et al. (2016) FEBS Journal 283: 3232-38, and Graham, D. , et al. Genome Biol. 2015; 16: 260.
  • gRNA comprises or alternatively consists essentially of, or yet further consists of a fusion polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA) ; or a polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA) .
  • a gRNA is synthetic (Kelley, M. et al. (201 ) J of Biotechnology 233 (2016) 74-83) .
  • "Homology” or "identity” or “similarity” can refer to sequence similarity between two peptides or between two nucleic acid molecules.
  • Homology can be determined by comparing a position in each sequence which can be aligned for purposes of comparison. For example, when a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or “non-homologous " sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the disclosure.
  • Homology refers to a percent (%) identity of a sequence to a reference sequence.
  • any particular sequence can be at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to any sequence described herein.
  • Whether such particular peptide, polypeptide or nucleic acid sequence has a particular identity/homology can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) .
  • the parameters can be set such that the percentage of identity is calculated over the full length of the reference sequence and that gaps in homology of up to 5% of the total reference sequence are allowed .
  • the identity between a reference sequence (query sequence, i . e . , a sequence of the disclosure) and a subject sequence, also referred to as a global sequence alignment can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990) ) .
  • the percent identity can be corrected by calculating the number of residues of the query sequence that are lateral to the N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
  • a determination of whether a residue is matched/aligned can be determined by results of the FASTDB sequence alignment. This percentage can be then subtracted from the percent identity, calculated by the FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score can be used for the purposes of this embodiment. In some cases, only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence are considered for this manual correction. For example, a 90 residue subject sequence can be aligned with a 100 residue query sequence to determine percent identity.
  • the deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity can be 90%.
  • a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query.
  • Hybridization can refer to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex can comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction can constitute a step in a more extensive process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC.
  • Examples of moderate hybridization conditions include: incubation temperatures of about 40 °C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC.
  • Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about 0.
  • lx SSC formamide concentrations of about 55% to about 75%
  • wash solutions of about lx SSC, 0. lx SSC, or deionized water.
  • hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
  • SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
  • the term "immune orthogonal” refers to a lack of immune cross-reactivity between two or more antigens.
  • the antigens are proteins (e.g. , Cas9) .
  • the antigens are viral antigens associated with a particular viral vector (e.g. , AAV) .
  • antigens typically include antigenic determinants having a particular sequence of 3 dimensional structure.
  • an antigenic determinant can comprise a domain or subsequence of a larger polypeptide or molecular sequence.
  • antigens that are immune orthogonal do not share an amino acid sequence of greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, greater than 10, greater than 11, greater than 12, greater than 13, greater than 14, greater than 15, or greater than 16 consecutive amino acids. In some embodiments, antigens that are immune orthogonal do not share any highly immunogenic peptides. In some embodiments, antigens that are immune orthogonal do not share affinity for a major histocompatibility complex (e.g. , MHC class I or class II) . Antigens that are immune orthogonal are amenable for sequential dosing to evade a host immune system.
  • MHC class I or class II major histocompatibility complex
  • immunosilent refers to an epitope or foreign peptide, polypeptide or protein that does not elicit an immune response from a host upon administration.
  • the peptide, polypeptide or protein does not elicit an adaptive immune response.
  • the peptide, polypeptide or protein does not elicit an innate immune response.
  • the peptide, polypeptide or protein does not elicit either an adaptive or an innate immune response.
  • an immunosilent peptide, polypeptide or protein has reduced immunogenicity.
  • isolated can refer to molecules or biologicals or cellular materials being substantially free from other materials.
  • the term “isolated” can refer to nucleic acid, such as DNA or RNA, or protein or polypeptide (e.g. , an antibody or derivative thereof) , or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source.
  • isolated also can refer to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an "isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and may not be found in the natural state.
  • the term “isolated” is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • isolated is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both cultured and engineered cells or tissues.
  • Messenger RNA or "mRNA” is a nucleic acid molecule that is transcribed from DNA and then processed to remove non-coding sections known as introns. In some cases, the resulting mRNA is exported from the nucleus (or another locus where the DNA is present) and translated into a protein.
  • pre-mRNA can refer to the strand prior to processing to remove non-coding sections. mRNA has "U” in place of "T” in cDNA coding sequences.
  • MHC Major Histocompatibility Complex
  • T- cells recognize antigenic peptides and trigger a cascade of events which leads to the destruction of pathogens and infected cells.
  • the MHC family is divided into three subgroups: class I, class II, and class III.
  • Class I MHC molecules have p2 subunits that are only recognized by CD8 co-receptors .
  • Class II MHC molecules have pl and p2 subunits that are only recognized by CD4 co-receptors. In this way MHC molecules chaperone which type of lymphocytes may bind to the given antigen with high affinity, since different lymphocytes express different T-Cell Receptor (TCR) co-receptors.
  • TCR T-Cell Receptor
  • MHC class I molecules bind short peptides, whose N- and C-terminal ends are anchored into pockets located at the ends of a peptide binding groove. While the majority of the peptides are nine amino acid residues in length, longer peptides can be accommodated by the bulging of their central portion, resulting in binding peptides of length 8 to 15. Peptides binding to class II proteins are not constrained in size and can vary from 11 to 30 amino acids long.
  • the peptide binding groove in the MHC class II molecules is open at both ends, which enables binding of peptides with relatively longer length.
  • the "core" refers to the amino acid residues that contribute the most to the recognition of the peptide. In some embodiments, the core is nine amino acids in length. In addition to the core, the flanking regions are also important for the specificity of the peptide to the MHC molecule.
  • ortholog is used in reference of another gene or protein and intends a homolog of said gene or protein that evolved from the same ancestral source or which are evolved artificially using molecular biology and genetic engineering. Orthologs may or may not retain the same function as the gene or protein to which they are orthologous .
  • Cas9 orthologs include S. aureus Cas9 ("spCas9”) , S. thermophiles Cas9, L. pneumophilia Cas9, N. lactamica Cas9, N. meningitides Cas9, B. longum Cas9, A. muciniphila Cas9, and O. laneus Cas9.
  • promoter refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters may be constitutive, inducible, repressible, or tissuespecific, for example.
  • a "promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
  • Non-limiting exemplary promoters include CMV promoter and U6 promoter.
  • protein protein
  • peptide and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics .
  • the subunits can be linked by peptide bonds. In another embodiment, the subunit can be linked by other bonds, e.g. , ester, ether, etc.
  • a protein or peptide can contain at least two amino acids and no limitation is placed on the maximum number of amino acids which can comprise a protein's or peptide's sequence.
  • amino acid can refer to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • fusion protein can refer to a protein comprised of domains from more than one naturally occurring or recombinantly produced protein, where generally each domain serves a different function.
  • linker can refer to a peptide fragment that is used to link these domains together - optionally to preserve the conformation of the fused protein domains and/or prevent unfavorable interactions between the fused protein domains which can compromise their respective functions.
  • polynucleotide and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown.
  • polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag) , exons, introns, messenger RNA (mRNA) , transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • the term also can refer to both double and single stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide can encompass both the double stranded form and each of two complementary single stranded forms known or predicted to make up the double stranded form.
  • polynucleotide sequence can be the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching .
  • polypeptide sequence can be the alphabetical representation of a polypeptide molecule.
  • This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional proteomics and homology searching.
  • recombinant expression system refers to a genetic construct or constructs for the expression of certain genetic material formed by recombination.
  • recombinant protein can refer to a polypeptide or peptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide or peptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous polypeptide or peptide.
  • sequencing can comprise bisulfite-free sequencing, bisulfite sequencing, TET-assisted bisulfite (TAB) sequencing, ACE-sequencing, high-throughput sequencing, Maxam-Gilbert sequencing, massively parallel signature sequencing, Polony sequencing, 454 pyrosequencing, Sanger sequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real time (SMRT) sequencing, nanopore sequencing, shot gun sequencing, RNA sequencing, Enigma sequencing, or any combination thereof.
  • TAB TET-assisted bisulfite
  • ACE-sequencing high-throughput sequencing
  • Maxam-Gilbert sequencing massively parallel signature sequencing
  • Polony sequencing 454 pyrosequencing
  • Sanger sequencing Illumina sequencing
  • SOLiD sequencing Ion Torrent semiconductor sequencing
  • DNA nanoball sequencing Heliscope single molecule sequencing
  • SMRT single molecule real time sequencing
  • nanopore sequencing shot gun sequencing
  • RNA sequencing En
  • the term "subject" is intended to mean any animal.
  • the subject may be a mammal; in further embodiments, the subject may be a bovine, equine, feline, murine, porcine, canine, human, or rat.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection (e.g. , using commercially available reagents such as, for example, LIPOFECTIN® (Invitrogen Corp. , San Diego, CA) , LIPOFECTAMINE® ( Invitrogen) , FUGENE® (Roche Applied Science, Basel, Switzerland) , JETPEITM (Polyplus-transfection Inc.
  • LIPOFECTIN® Invitrogen Corp. , San Diego, CA
  • LIPOFECTAMINE® Invitrogen
  • FUGENE® Roche Applied Science, Basel, Switzerland
  • JETPEITM Polyplus-transfection Inc.
  • treat refers to ameliorating symptoms associated with a disease or disorder (e.g. , cancer, Covid-19 etc. ) , including preventing or delaying the onset of the disease or disorder symptoms, and/or lessening the severity or frequency of symptoms of the disease or disorder .
  • a disease or disorder e.g. , cancer, Covid-19 etc.
  • vector can refer to a nucleic acid construct deigned for transfer between different hosts, including but not limited to a plasmid, a virus, a cosmid, a phage, a BAG, a YAC, etc.
  • a "viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • plasmid vectors can be prepared from commercially available vectors.
  • viral vectors can be produced from baculoviruses , retroviruses, adenoviruses, AAVs, etc. according to techniques known in the art.
  • the viral vector is a lentiviral vector.
  • viral vectors examples include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.
  • Infectious tobacco mosaic virus (TMV) -based vectors can be used to manufacturer proteins and have been reported to express Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106 (15) : 6099-6104) .
  • Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5: 434-439 and Ying et al.
  • a vector construct can refer to the polynucleotide comprising the retroviral genome or part thereof, and a gene of interest. Further details as to modern methods of vectors for use in gene transfer can be found in, for example, Kotterman et al. (2015) Viral Vectors for Gene Therapy: Translational and Clinical Outlook Annual Review of Biomedical Engineering 17. Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art.
  • Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif. ) and Promega Biotech (Madison, Wis. ) .
  • the promoter is a pol III promoter.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. , bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) .
  • Other vectors e.g. , non-episomal mammalian vectors
  • vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors.”
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and 'Vector” can be used interchangeably.
  • the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g. , replication defective retroviruses, adenoviruses and adeno-associated viruses) , which serve equivalent functions.
  • the vector or plasmid contains sequences directing transcription and translation of a relevant gene or genes, a selectable marker, and sequences allowing autonomous replication or chromosomal integration.
  • Suitable vectors comprise a region 5' of the gene which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcription termination. Both control regions may be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions may also be derived from genes that are not native to the species chosen as a production host.
  • the vector or plasmid contains sequences directing transcription and translation of a gene fragment, a selectable marker, and sequences allowing autonomous replication or chromosomal integration.
  • Suitable vectors comprise a region 5' of the gene which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcription termination. Both control regions may be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions may also be derived from genes that are not native to the species chosen as a production host.
  • Initiation control regions or promoters which are useful to drive expression of the relevant pathway coding regions in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genetic elements is suitable for the present invention including, but not limited to, lac, ara, tet, trp, IPL, IPR, T7, tac, and trc (useful for expression in Escherichia coli and Pseudomonas) ; the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus subtilis , and Bacillus licheniformis; nisA (useful for expression in gram positive bacteria, Eichenbaum et al.
  • Termination control regions may also be derived from various genes native to the preferred hosts .
  • Immunogenicity is a major concern for protein-based therapeutics, particularly those derived from non-human species. Induction of the immune response can render treatments ineffective and cause serious, even life-threatening side effects. One strategy to overcome this issue is to mutate particularly immunogenic epitopes in the therapeutic target.
  • Another problem is that reading out combinatorial mutations scattered across large (>lkb) regions of the protein is extremely difficult using short read sequencing.
  • Using short barcodes attached to each variant to genotype libraries post-screen has proved effective but is limited by the difficulty of constructing large combinatorial libraries in which each member has a short, unique barcode.
  • the disclosure provides a 3-part strategy to overcome library size constraints in both the number of unique members and the length of the mutagenized region.
  • the disclosure provides a protein engineering platform capable of screening millions of combinatorial variants simultaneously with mutations spread across the full length of arbitrarily large proteins, with computation- guided mutation design to maximize the probability of exploring functional mutation space (FIG. 1A) .
  • the platform can be applied iteratively to tackle particularly challenging engineering tasks by exploring huge swaths of combinatorial mutation space unapproachable using previous techniques.
  • target regions were selected within the protein of interest using software which predicts HLA-binding and peptide immunogenicity. It can be difficult to functionalize these predictions, however, because HLA loci are highly polymorphic, and each HLA allele will have its own particular ligand binding profile.
  • an approximation of global HLA allele frequencies was created using data from the Allele Frequency Net Database. These frequencies were used to scale immunogenicity predictions such that the top hits are the peptides likely to be the most immunogenic epitopes for the largest number of people globally.
  • the method comprises, consists of, or consists essentially of identifying targeted regions of a protein associated with HLA binding.
  • the targeted regions can be ranked by HLA allele frequency using data from the Allele Frequency Net Database ( [www . ] allelef requencies . net ; brackets provided to eliminate hyperlinks) .
  • the frequencies are used for immunogenicity predictions, such that the top hits are the peptides likely to be the most immunogenic epitopes for the largest number of people globally.
  • mutational variants are narrowed by identifying mutations that have the least disruption to protein function.
  • the disclosure provides a method for engineering a protein or virus to be less immunoreactive, comprising one or more of the following steps: identifying target regions of the DNA sequence of protein or virus that are predicted to have human leukocyte antigen (HLA) -binding and/or peptide immunogenicity; identifying single nucleotide polymorphisms (SNPs) or mutations in the targeted region and other regions that are not deleterious to the functioning of the protein; screening a library assembled using standard synthesis and assembly methods by applying the above identifying criteria to find functional variants of the protein or virus; sequencing the functional variants of the protein or virus; and/or mapping genotype to phenotype from the sequences of the functional variants to identify variant candidates that are likely functionally active and have mutations that result in the protein or virus exhibiting less immunogenicity.
  • HLA human leukocyte antigen
  • SNPs single nucleotide polymorphisms
  • Non-limiting exemplary proteins of interest include cytidine deaminases, which can be used for gene editing via catalysis of DNA base change from C to T (e.g. APOBEC - conserveed across many species e.g.
  • ZFNs Zing Finger nucleases
  • TALENs transcriptional activatorlike effector nucleases
  • TALENs transcriptional activatorlike effector nucleases
  • ZFNs and TALENs The domains of the site-specific enzymes mentioned above (ZFNs and TALENs) are well characterized and subject of extensive engineering to generate the desired specificity. Thus, many variants exist of such proteins. Additional proteins for which HLA-binding affinity analysis is relevant include Cas9 proteins and AAV capsids, both of which are used in CRISPR based gene editing.
  • the methods disclosed herein provide for reducing the immunogenicity of a CRISPR associated protein.
  • CRISPR associated proteins include, but are not limited to, Cas9, Casl2, Casl3, Casl4.
  • the CRISPR associated protein is a Cas9.
  • the Cas9 is Streptococcus pyogenes Cas9 (SpCas9) .
  • the Cas9 proteins the orthologs are selected from S. pyogenes Cas9 (spCas9) , S. aureus Cas9 (saCas9) , B. longum Cas9, A. muiciniphilia Cas9, or O.
  • the disclosure provides methods to "humanize" the CRISPR associated protein by swapping high immunogenic domains or peptides with less immunogenic counterparts. This is particularly useful to enable the application of CRIPSR based therapeutics for repeat treatments.
  • the disclosure teaches methods and methodology to screen mutations in selected targeted regions of proteins, such as CRISPR associated proteins, in order to reduce immunogenicity.
  • embodiments of the disclosure relate to a modified CRISPR associated protein that has lower immunogenicity to promote immune evasion.
  • the modified proteins can replace existing wildtype proteins for any application requiring in vivo delivery, which would potentially have no loss of efficacy after repetitive use.
  • isolated polynucleotides encoding a modified Cas9 protein, wherein the modified Cas9 comprises, consists of, or consists essentially of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, or twenty or more of the amino acid modifications in targeted regions to lower the immunogenicity of the protein.
  • the disclosure provides an engineered immune- silenced Cas9 comprising a sequence of SEQ ID NO: 2 and having one or more mutations selected from the group consisting of: P28L, L237C, Y286Q, S318 (H/C) , S368C, F498T, L514 (T/G) , L616G, L623Q, L636D, F704A, L727 (P/G) , L816D, Y1016 (K/G) , L1245G, I1273Q, L1282 (A/E) , and/or Y1294Q.
  • vectors comprising an isolated polynucleotide encoding an engineered Cas9 comprising one or more mutations selected from the group consisting of: P28L, L237C, Y286Q, S318 (H/C) , S368C, F498T, L514 (T/G) , L616G, L623Q, L636D, F704A, L727 (P/G) , L816D, Y1016 (K/G) , L1245G, I1273Q, L1282 (A/E) , and/or Y1294Q.
  • the vector is an AAV vector, optionally wherein the AAV vector is AAV5.
  • the disclosure provides an isolated polypeptide comprising (i) SEQ ID NO: 2 having a mutation (s) selected from P28L, L237C, Y286Q, S318 (H/C) , S368C, F498T, L514 (T/G) , L616G, L623Q, L636D, F704A, L727 (P/G) , L816D, Y1016 (K/G) , L1245G, I1273Q, L1282 (A/E) , Y1294Q, and any combination thereof, wherein the polypeptide has Cas9 activity and wherein the polypeptide has reduced immunogenicity compared to SEQ ID NO: 2 lacking any one or more of the mutations; (ii) a sequence that is 95%-99% identical to (i) ; and (iii) related homolog/orthologs having mutations corresponding to the mutations in SEQ ID NO: 2 and having Cas9 activity.
  • the disclosure also provided isolated polynucleotides encoding the polypeptide of (i) - (iii) above.
  • the polynucleotides can be RNA or DNA.
  • the polynucleotides can be cloned into a vector for expression and/or delivery to a subject.
  • the targeted regions to lower the immunogenicity of the protein are identified using a model that predicts human leukocyte antigen (HLA) -binding and peptide immunogenicity.
  • Models for determining HLA-binding affinity and peptide immunogenicity are likewise known in the art and may include computational methods available through software or publicly accessible databases or "wet lab” assays. Examples of computational methods of predicting HLA-binding affinity include, but are not limited to, the MHC prediction models available through the IEDB Analysis Resource ( [http : //] tools . immuneepitope .
  • HLA-binding can be determined or computational predictions thereof can be validated using assays, such as, but not limited to, immunoassays, such as ELISA, microarray, tetramer assay, and peptide-induced MHC stabilization assay.
  • assays such as, but not limited to, immunoassays, such as ELISA, microarray, tetramer assay, and peptide-induced MHC stabilization assay.
  • modifications in the proteins can be optimized to reduce the immunogenicity of the protein when administered to a particular subject or patient.
  • the comparisons can be host-restricted, such that the protein is optimized to reduce the immunogenicity of the protein when administered to a particular host, e.g. , a mouse or a human.
  • SNPs or mutations are identified by using phylogenetic methods to scan natural variation among naturally occurring proteins, mutations generated in the course of research and engineering efforts, and protein orthologs from closely related species.
  • the SNPS or mutations are identified, or further identified, by using immunological prediction of candidate mutations to ensure significant loss of immunogenicity within the targeted region in order to preserve function while reducing immunogenicity.
  • the disclosure contemplates use of the methods of the disclosure for reducing the immunogenicity of viruses. The methods can be applied to a variety of types of viruses that present a risk of eliciting an immune response, particularly those used in gene therapy or gene delivery.
  • viruses include but are not limited to, retroviruses, adenoviruses, adeno-associated viruses (AAVs) , alphaviruses, lentiviruses , pox viruses, and herpes viruses.
  • the virus is an AAV.
  • the AAV is selected from AAV1, AAV2, AAV5, AAV6, AAV7, and AAV8.
  • the AAV is AAV5.
  • the disclosure provides methods encompassing a step of identifying target regions of proteins and the corresponding polynucleotide coding sequence of a virus that are predicted to have human leukocyte antigen (HLA) -binding and/or peptide immunogenicity target regions.
  • the targeted regions are identified by aligning conserved sequence regions across AAV serotypes.
  • the SNPs are identified by aligning the sequences of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more AAV variants that have been sequenced from natural or engineered sources.
  • the SNPs are located in the target regions.
  • long-read sequencing technologies capable of sequencing the entire sequence of each protein or viral variant in one sequencing reaction are used.
  • the long-read sequencing technologies is capable of generating 10-15 Gb of sequencing reads per run.
  • sequencing technologies include, but are not limited to, Oxford Nanopore's Minion sequencer.
  • a method disclosed herein encompasses the step of evaluating the immunoreactivity of variant candidates in one or more immunoassays.
  • the one or more immunoassays comprise detecting the presence of antibodies to the variant candidates (AVA antibodies) , when the variant candidates are administered in vivo to an animal.
  • the one or more immunoassays comprise an enzyme-linked immunosorbent assays (ELISAs) , electrochemiluminescence (ECL) assays and/or antigen-binding tests, wherein the one or more immunoassays utilize AVA antibodies.
  • compositions used in various therapies wherein the composition comprises a potentially immunogenic molecule such as a protein or polypeptide.
  • the methods of the disclosure can be used to identify domains that are immunogenic and identify mutations that reduce immunogenicity.
  • Cas9 is a protein used in gene editing in vivo, but has been shown to have immunogenic potential.
  • the disclosure provides for a polynucleotide having at least 90%, 95%, 97%, 98%, or 99% sequence identity to the sequence presented in SEQ ID NO: 1, wherein the polnucleotide encodes a polypeptide having at least 90% identity to SEQ IDNO:2 having reduced immunogenicity and wherein the protein has Cas9 like activity.
  • the disclosure also provides for a protein having a polypeptide sequence that has at least 90%, 95%, 97%, 98%, or 99% sequence identity to the sequence presented in SEQ ID NO: 2, wherein the protein has Cas9 like activity.
  • the protein has less immunogenicity than Cas9.
  • the disclosure further provides for a CRISPR-Cas9 system comprising a protein disclose herein that has Cas9 like activity.
  • the CRISPR-Cas9 system further comprises RNA that comprise a shot sequence that binds to a specific target sequence of DNA in a genome.
  • RNAs can be generated for target specificity to target a specific gene, optionally a gene associated with a disease, disorder, or condition.
  • the guide RNAs facilitate the target specificity of the CRISPR/Cas9 system. Further aspects such as promoter choice, may provide additional mechanisms of achieving target specificity - e.g. , selecting a promoter for the guide RNA encoding polynucleotide that facilitates expression in a particular organ or tissue. Accordingly, the selection of suitable RNAs for the particular disease, disorder, or condition is contemplated herein .
  • the disclosure provides for a virus encoded by a polynucleotide sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to the sequence presented in Figure 5, wherein the virus has AAV5 like activity.
  • the disclosure also provides for a virus capsid having at least 90%, 95%, 97%, 98%, or 99% sequence identity to the sequence presented in SEQ ID NO: 50, wherein the virus has AAV5 like activity.
  • the virus has less immunogenicity than AAV5.
  • the disclosure also provides for an AAV system comprising a virus disclosed herein that has AAV5 like activity.
  • the AAV system is used for gene therapy.
  • Administration of the AAV variant or compositions thereof can be affected in one dose, continuously or intermittently throughout the course of treatment. Administration may be through any suitable mode of administration, including but not limited to: intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular , epidural, intracerebral, intracerebroventricular , sub-retinal, intravitreal , intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmuccosal , and inhalation.
  • Methods of determining the most effective route and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. It is noted that dosage may be impacted by the route of administration. Suitable dosage formulations and methods of administering the agents are known in the art. Non-limiting examples of such suitable dosages may be as low as 1E+9 vector genomes to as much as 1E+17 vector genomes per administration .
  • a modified virus and compositions of the disclosure having reduced immunogenicity can be administered in combination with other treatments, e.g. those approved treatments suitable for the particular disease, disorder, or condition.
  • Doses suitable for uses herein may be delivered via any suitable route, e.g. intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods, and/or via single or multiple doses. It is appreciated that actual dosage can vary depending on the recombinant expression system used (e.g. AAV or lentivirus) , the target cell, organ, or tissue, the subject, as well as the degree of effect sought. Size and weight of the tissue, organ, and/or patient can also affect dosing. Doses may further include additional agents, including but not limited to a carrier.
  • a carrier e.g. intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods, and/or via single or multiple doses. It is appreciated that actual dosage can vary depending on the recombinant expression system used (e.g. AAV or lentivirus) , the target cell, organ, or tissue, the subject, as well as the degree of effect sought. Size and weight of the
  • Non-limiting examples of suitable carriers are known in the art: for example, water, saline, ethanol, glycerol, lactose, sucrose, dextran, agar, pectin, plant-derived oils, phosphate-buf fered saline, and/or diluents. Additional materials, include those disclosed in paragraph [00533] of WO 2017/070605 may be used with the compositions disclosed herein. Paragraphs [00534] through [00537] of WO 2017/070605 also provide non-limiting examples of dosing conventions for CRISPR-Cas systems which can be used herein. In general, dosing considerations are well understood by those in the art .
  • compositions or kits comprising any one or more of the variant proteins and/or variant viruses described herein.
  • the carrier is a pharmaceutically acceptable carrier.
  • compositions of the present invention may comprise a variant Cas9 described herein or a polynucleotide encoding said Cas9, optionally comprising a variant AAV described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g.
  • compositions of the present disclosure may be formulated for oral, intravenous, topical, enteral, and/or parenteral administration. In certain embodiments, the compositions of the present disclosure are formulated for intravenous administration.
  • a library was constructed in a stepwise process.
  • the full-length gene was broken up into short blocks of no more than 1000 bp, which overlap by 30 bp on each end.
  • Each block is designed such that it contains no more than 3 or 4 target epitopes to mutagenize.
  • block 2 of the SpCas9 library contains 3 target epitopes (see, e.g. , FIG. 2B) .
  • One variant mutation was designed to test at two of these sites, and two variant mutations at the final site.
  • each of these block mixes were assembled into a fully combinatorial library of Cas9 sequences using a two- step PGR assembly method.
  • the first annealing-extension step allows each of the blocks to anneal to and prime their attachment to neighboring blocks using their 30 bp overlapping ends.
  • DNA polymerase then extends these attached fragments into full length Cas9 genes with the order of block assembly being specified by the unique 30 bp overhangs at each block junction.
  • primers binding to only the far 3' and 5' ends of the Cas9 sequence are used to amplify the full length Cas9 library in a standard PCR reaction.
  • the library is then purified and inserted into an appropriate cloning/expression vector via Gibson assembly.
  • nanopore sequencing was applied using the Oxford Nanopore (ONT) MinlON platform.
  • ONT Oxford Nanopore
  • PCR amplification was performed on the full-length Cas9 gene from the plasmid library preparation and the nanopore sequencing adapters were ligated as per standard ONT protocol.
  • a IX sequencing depth of the library was performed, which was sufficient to serve as a QC check and to ensure library diversity.
  • the low sequencing depth and noisy nature of nanopore reads only allowed reliable identification of 304, 060 unique library elements. The read count distribution suggests that this is an under-sampling of the library diversity, and the mutation density distribution, i . e .
  • HPRT1 hypoxanthine phosphorribosyltransferase 1
  • HeLa cells were transduced with lentivirus particles containing wild-type Cas9 and either a HPRTl-targeting guide RNA (gRNA) or a nontargeting guide. After selection with puromycin, cells were treated with 6TG concentrations ranging from 0-14 pg/mL for one week. Cells were stained with crystal violet at the end of the experiment and imaged. 6 pg/mL was selected as all cells containing non-targeting guide had died while cells containing the HPRT1 guide remained viable ( Figure 6) .
  • gRNA HPRTl-targeting guide RNA
  • HeLa cells were transduced with lentiviral particles containing variant library or wild-type Cas9 along with the HPRTl-targeting gRNA at 0.3 MOI and at greater than 75-fold coverage of the library elements.
  • Cells were selected using puromycin after two days and 6TG was added once cells reached 75% confluency.
  • genomic DNA was extracted from remaining cells and full-length Cas9 sequences were PCR amplified.
  • Nanopore-compatible sequencing libraries were prepared per manufacturer's instructions and sequenced on the MinlON platform. This screening procedure was performed in two replicates.
  • GFP expression can be restored via homologous recombination, where the DNA is edited via a guide that targets the AAVS1 locus fragment and repaired with a GFP donor sequence.
  • GFP+ cells can then be quantified by flow-activated cell sorting (FACS) , providing information on editing efficiency.
  • FACS flow-activated cell sorting
  • V- 20 variants were constructed as detailed in Table A.
  • a large majority of the variants were capable of editing, providing further confidence in the network we constructed, and in particular highlighted variants V8 and V12 with high editing capability and 8 and 7 mutations respectively (FIGs. 11 and 12) .
  • Cas9 variants were generated with multiple top immunogenic epitopes simultaneously de-immunized while retaining editing functionality.
  • Table B Top immunogenic epitopes across Cas9 orthologs (Mutational reference amino acids exclude position 1 methionine, e.g. , P27L excludes methionine at position 1 of SEQ ID NO: 2 Sp-Cas9; or alternatively stated, methionine is amino acid "0”) .
  • T-regi topes Inserting T-regi topes .
  • a further de-immuni zation approach beyond the mutation or deletion of MHC-binding cores is to inhibit immune activation at the level of cell s ignaling .
  • Paramount in facilitating this proces s is the induction of inhibitory T-reg cells which modulate the activity of other T-cells to promote tolerance of foreign antigens .
  • One canonical pathway in which T-reg induction helps to facilitate tolerance of antigenic divers ity is in the potential adaptive response to antibodies themselves . As antibodies are highly polymorphic and undergo substantial mutational remodeling during the proces s of B-cell activation and maturation . As a result , it might be expected that these neo-antigens may create an adaptive immune response .
  • T-regitopes are recogni zed speci fically by regulatory T-cells to promote a tolerogenic response to proteins bearing these T-regitopes .
  • these immune-modulating sequences can be utili zed in the context of foreign protein therapeutics to dampen or avoid a problematic immune response .
  • Table D Immunogenic and conserved AAV-VP1 epitopes and de-immunizing mutations (methionine is position "0")
  • the capsid sequences of each ortholog were chemically synthesized and split into two blocks from the 5' and 3' end.
  • a process similar to the one used for assembling the Cas9 variants was done.
  • the first step involves the annealing-extension step followed by the addition of primers to amplify the full length ortholog.
  • the full length capsid sequences see, e.g. , SEQ ID NO:2-48, which include both polynucleotide coding sequences and polypeptide sequence
  • SEQ ID NO:2-48 which include both polynucleotide coding sequences and polypeptide sequence
  • a triple transfection method was used for AAV production using HEK 293T cells and purified with an iodixanol gradient.
  • Cells were transfected once the confluence reached between 70% and 90% in a 5x15 cm 2 plate. Plates were transfected with 10pg of the plasmid with full length inserted ortholog capsid sequence or pxR-5 used as a control, 10pg of transfer vector, and 10pg of pAd4 helper vector.
  • the virus was collected after 72 hours and purified using an iodixanol-density- gradient ultracentrifugation method. After dialysis and filtration, the virus was quantified by qPCR.
  • Results of viral formation were analyzed by comparing titers of the AAV orthologs to AAV5 titers used as a control. The viral formation of 14 AAV orthologs were tested and have demonstrated 8 AAV orthologs successfully packaging and producing virus (FIG. 15) .

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Abstract

L'invention concerne des méthodes d'ingénierie de protéines et de virus pour réduire leur immunogénicité, des protéines et des virus obtenus par lesdites méthodes, comprenant des protéines présentant une activité de type Cas9 et des virus présentant une activité de type AAV5.
PCT/US2021/061682 2020-12-02 2021-12-02 Ingénierie d'aav orthogonal immunitaire et de furtif immunitaire crispr-cas Ceased WO2022120103A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023034997A1 (fr) * 2021-09-03 2023-03-09 Biomarin Pharmaceutical Inc. Compositions capsidiques de vaa et méthodes d'administration
WO2023034990A1 (fr) * 2021-09-03 2023-03-09 Biomarin Pharmaceutical Inc. Compositions capsidiques de vaa et méthodes d'administration
WO2025171041A1 (fr) * 2024-02-05 2025-08-14 The General Hospital Corporation Protéines casphi2 (cas12j-2) modifiées

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017081288A1 (fr) * 2015-11-11 2017-05-18 Lonza Ltd Protéines associées à crispr (cas) à immunogénicité réduite
WO2019180243A1 (fr) * 2018-03-22 2019-09-26 Charité-Universitätsmedizin Berlin Immunité médiée par les lymphocytes t réactifs vis-à-vis d'une protéine associée à crispr

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017081288A1 (fr) * 2015-11-11 2017-05-18 Lonza Ltd Protéines associées à crispr (cas) à immunogénicité réduite
WO2019180243A1 (fr) * 2018-03-22 2019-09-26 Charité-Universitätsmedizin Berlin Immunité médiée par les lymphocytes t réactifs vis-à-vis d'une protéine associée à crispr

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP4256046A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023034997A1 (fr) * 2021-09-03 2023-03-09 Biomarin Pharmaceutical Inc. Compositions capsidiques de vaa et méthodes d'administration
WO2023034990A1 (fr) * 2021-09-03 2023-03-09 Biomarin Pharmaceutical Inc. Compositions capsidiques de vaa et méthodes d'administration
WO2025171041A1 (fr) * 2024-02-05 2025-08-14 The General Hospital Corporation Protéines casphi2 (cas12j-2) modifiées

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