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WO2011038187A1 - Diversification de virus adéno-associé (aav) commandé et bibliothèques préparées à partir de ce dernier - Google Patents

Diversification de virus adéno-associé (aav) commandé et bibliothèques préparées à partir de ce dernier Download PDF

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WO2011038187A1
WO2011038187A1 PCT/US2010/050135 US2010050135W WO2011038187A1 WO 2011038187 A1 WO2011038187 A1 WO 2011038187A1 US 2010050135 W US2010050135 W US 2010050135W WO 2011038187 A1 WO2011038187 A1 WO 2011038187A1
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roi
aav
primer
subdomains
sequence
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James M. Wilson
Luc H. Vandenberghe
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University of Pennsylvania Penn
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—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

  • AAV adeno-associated viruses
  • directed evolution involves the so-called directed evolution which may be performed in a high through-put approach [N. Maheshri, et al, "Directed evolution of adeno-associated virus yields enhanced gene delivery vectors", Nature Biotechnology, 24 (2): 198-204 (February 2006); J. T. Koerber, et al, Construction of diverse adeno- associated viral libraries for directed evolution of enhanced gene delivery vehicles", Nature Protocols, 1(2): 701-706 (2006)].
  • This method involves diversifying AAV capsid (cap) genes through error-prone polymerase chain reaction (PCR) [Maheshri et al, cited above and Koerber et al, cited above] followed by the staggered extension process [H.
  • Fig 1 provides a cartoon example of a two step method to generate diversity within delineated domains of coding sequence (CDS).
  • CDS delineated domains of coding sequence
  • two regions of interest (ROIs) are defined within a CDS.
  • the ROI are delineated by primers according to the method in a manner that all eventual permutations of the ROI universally link up with the flanking regions (FRs).
  • Diversity is generated by any number of methods in any number of combinations e.g. one ROI be amplified from different templates without or without different primers or pools of primers whereas another ROI undergoes a DNA polymerization or ligation step that introduces variation through shuffling or mutagenesis.
  • the entire CDS is amplified by SOE PCR that then splices all individual components in a correct order due to the complementarity of the primers.
  • N number of ROI
  • EP Error Prone
  • STEP Staggered extension process
  • ⁇ different.
  • Fig. 2A is a Neighbor-Joining dendrogram of the AAV capsid illustrating rh32.33 structural distinctness from all serotypes with AAV4 the most homologous capsid.
  • Fig. 2B provides various quantitative measures of AAV8 and rh32.33 neutralization by human serum including IVIG neutralization in the in vitro NAB assay (titer represents dilution of 50% transduction inhibition), estimate of seroprevalence in 888 individuals in worldwide populations (Europe, US, Africa and Australia) and lowest IVIG dose at which statistically significant in vivo transduction inhibition is observed at a dose of 10 n GC per mouse.
  • Fig. 3 A is a cartoon showing the viral genome backbones for pAAV8 and the pAAVivo - 8.
  • Fig. 3B is a cartoon showing the AAVrh32.33 capsid open reading frame (ORF) broken down into preliminary subdomains which consist of either constant (C) or hypervariable (H) domains.
  • Constant (C) regions CI , C2, C3, C4, C5, C6, C7 and C8 are illustrated, as are hypervariable (H) regions HI, H2, H3, H4, H5, H6, H7 and H9.
  • Fig. 3C provides a description of the first cycle polymerase chain reaction (PCR) in the method of the invention.
  • Individual domains here H are amplified flanked by oligonucleotide mixtures with overlapping sequences to flanking domains with complementarity to either the same or a different serotype.
  • Fig. 3D provides a schematic of 2 nd round PCR of a full capsid open reading frame with only individual subdomains as template and yielding pAAV and pAAVivo compatible BgHI-Spel flanked capsids.
  • Fig. 4 is a bar chart comparing the ability of vectors containing mutant AAV8 capsid proteins, generated using an AAV combinatorial library approach of the invention, to express a marker gene product (enhanced green fluorescent protein) following incubation with different monoclonal antibodies, including two an-AAV8 antibodies.
  • a marker gene product enhanced green fluorescent protein
  • the method of the invention permits the generation of novel proteins and protein coding sequences, while providing the ability to control where diversity is introduced and retaining regions of interest within the protein where no changes are introduced.
  • the method of invention permits the number of nonfunctional proteins to be reduced by permitting one to define regions which are critical to the desired function. This allows for a more representative, functional combinatorial library of a size manageable within the context of a bacterial library (about 10 7 molecules).
  • a method for the directed production of a combinatorial library of an altered protein coding sequence.
  • at least a first region of interest (ROI) and at least a second ROI within a protein coding sequence are predetermined and at least a first primer set which is specific for at least a first subdomain containing the at least first ROI and at least a second primer set which is specific for at least second subdomain containing the at least second ROI are provided.
  • ROI region of interest
  • second primer set which is specific for at least second subdomain containing the at least second ROI
  • the subdomains are specifically amplified with a series of primer sets which comprises the at least first primer set and the at least second primer set, wherein each of the primer sets each consist of a right primer and a left primer which are complementary to a junction located between consecutive fragments in the protein coding sequence, wherein a predetermined subset of the amplified subdomains containing the at least first ROI and/or the at least second ROI are subject to different conditions in order to generate a subset of subdomains with altered sequences in the at least first ROI and/or the at least second ROI.
  • the resulting amplified subdomains are directionally and positionally assembled to generate a combinatorial library of altered full-length protein coding sequences.
  • the invention provides a method for the directed production of a combinatorial library of an altered adeno-associated virus gene.
  • the method may utilize a single AAV gene sequence in which at least two regions of interest (ROI) have been identified.
  • ROI regions of interest
  • the method may utilize two or more different AAVs for preparation of the directed combinatorial library.
  • At least a first primer set which specifically amplifies one of the at least two ROI is designed.
  • the primer set consists of a right primer (P R i) and left primer (Pu) > wherein one of the P R or P L has 5' complementarity to nucleotide sequences in the sequence the first ROI and the other has 3' complementary to the first ROI.
  • at least a second primer set which specifically amplifies a second of at least two ROI wherein said second primer set consists of a right primer (PR2) and left primer (Pu), wherein one of the Pm or PL2 has 5' complementarity to nucleotide sequences in the sequence flanking the second ROI and the other has 3' complementary to the second ROI, is provided.
  • primer sets are used to specific amplify the ROI (templates) to which they are directed and thereby generate a series of building blocks which correspond to subdomains within the full-length gene sequence, which subdomains, when assembled, span the full length AAV gene sequence.
  • the method permits a subset of the building blocks containing altered sequences to be generated from the at least one of the first or second ROI to form a plurality of diverse building blocks corresponding to the first or second ROI.
  • the resulting amplified regions (building blocks) are directionally and positionally assembled to generate a combinatorial library of full-length AAV genes, each of which contains the at least one ROI with altered sequences.
  • the invention provides a combinatorial library produced using the method of the invention.
  • this method provides a collection or library of sequences which contains a number of variant sequences, which variants are unknown in nature.
  • the present invention provides a method for engineering DNA and/or proteins in defined regions of biological relevance in a combinatorial manner.
  • One advantage of this invention lies is that variability can be introduced in a specific manner in regions of interest in a combinatorial fashion while preserving domains that one chooses not to perturb.
  • a method for the directed production of a combinatorial library of an altered protein coding sequence involves predetermining at least two different nucleotide sequence fragments (regions) which are of interest (ROI) in which the sequence diversity is to be introduced in a manner that may be different for the different subdomains.
  • the full-length protein coding sequence is divided into predetermined subdomains which are fragments spanning the full-length protein coding sequence.
  • a protein coding sequence is an open reading frame (ORF) for a selected protein or polypeptide.
  • a fragment of the protein coding sequence refers to a nucleic acid sequence which is a discrete portion of the protein coding sequence, which is shorter in length than the full-length protein coding sequence. Such a fragment may be internal, or located at the 5' or 3 ' terminus of the protein coding sequence. In one embodiment, the fragment is at least about 15 base pairs in length, and contains at least 15 base pairs less than the full-length open reading frame, preferably more.
  • the protein coding sequence is divided into "subdomains" which are nucleic acid fragments of the protein coding sequence used to generate a combinatorial library.
  • the entire protein coding sequence is divided into contiguous subdomains such that the full-length protein coding sequence is represented.
  • the location where two contiguous subdomains meet is termed herein a junction.
  • a protein coding sequence or a portion thereof may be divided into more than one set of subdomains.
  • each subdomain is large enough to accommodate a primer for PCR isolation, e.g., at least 15, 25, 30, or 50 nucleotides in length and may be as large a fragment as can be amplified via PCR.
  • a subdomain may contain a ROI.
  • a subdomain may contain no sequences other than the ROI.
  • a subdomain may contain nucleic acids which are 5 ! and/or 3 ! to the ROI.
  • a protein coding sequence is divided into at least one set of subdomains. In one embodiment, a protein coding sequence is divided into two or more sets of subdomains. It follows then, that the method of the invention may utilize multiple subdomains of different length which span the coding sequence.
  • the preparation of a library according to the present invention involves the use of two or more subdomains containing the same ROI (i.e., different ROI templates). Since one of the techniques for modifying polymerase chain reactions of a ROI involves varying the template, it will be understood that there can be multiple subdomains which contain the same ROI.
  • region of interest refers to a nucleic acid fragment within a protein coding sequence that has been pre-selected to be either altered or changed by some means, or to remain unchanged or unperturbed.
  • an ROI is at least 15 nucleotides in length and may be up to 95% of the length of the protein coding sequence.
  • An ROI may be about 15 nucleotides to 25 nucleotides to about 60% of the length of the protein coding sequence. In one embodiment, the ROI may be about 50 base pairs to about 800bp. However, one can readily select design shorter or longer ROI lengths.
  • the ROI is pre-selected based upon the function which is performed by the peptide or polypeptide for which the ROI codes.
  • one of skill in the art may pre-select the coding sequence corresponding to a specific protein structural element to remain unchanged and thus identify the coding sequence as an ROI.
  • one of skill in the art may select a hypervariable region of a viral capsid to a ROI, which is to be targeted for sequence alteration, or to be swapped with another hypervariable region, e.g., from another source or from a non-contiguous region within the same virus source.
  • an ROI within an AAV capsid, one may designate one or more conserved regions as an ROI, or pre-select one or more regions associated with AAV capsid functional variation (variable regions V, VIII and/or IX), transduction efficiency (variable regions I, II, ⁇ , ⁇ , V, VI and/or IX), and antigenic recognition (variable regions I, III, IV, V, VI, VII, VIII, IX,) as an ROI.
  • an ROI may be selected from receptor binding sites, DNA binding regions, phospholipase activity regions, or other desired domains.
  • an ROI may also be pre-selected based upon other considerations.
  • altered ROIs and/or altered subdomains are sequences in which nucleic acid bases have been changed as compared to the ROI and/or subdomain derived from its respective protein coding sequence.
  • these changes are introduced by changes in polymerase chain reaction conditions such as are described herein and/or which are know to those of skill in the art.
  • These different PCR conditions may include, e.g., varying the primer (e.g. changing the length of the primer, altering the sequences of the primer), varying the templates (e.g., changing the location of the junction between the ROI), varying the dNTP concentrations, and varying the salt concentrations and/or buffer conditions.
  • introducing diversity which covers introduction of diversity by any means.
  • the method of the invention permits one to predetermine which ROIs and/or subdomains are unperturbed or unaltered.
  • altered protein coding sequences includes protein coding sequences which comprise altered ROIs and/or altered subdomains as defined in the prior paragraph. "Altered protein coding sequences” also include sequences which are hybrids or chimera formed by recombining subdomains from at least a second protein coding sequence with those of a first protein coding sequence. Such "altered protein coding sequences” may contain subdomains which are derived from altered ROIs and/or altered subdomains from the same or a different protein coding sequence source and/or and unaltered sequences from at least one other protein coding sequence source. Thus, altered protein coding sequences may encode chimeric or hybrid proteins, optionally with altered ROI(s) and/or altered subdomain(s).
  • altered proteins are the products encoded by the "altered protein coding sequences" as defined herein. Such altered proteins may include proteins, polypeptides, enzymes, viral capsids, viral capsid proteins, viral envelopes, viral envelope proteins, polypeptides, or other products.
  • the term "unperturbed” or “conserved” ROI and/or subdomains refers to sequences in which nucleic acid bases remain unchanged as to the ROI and/or subdomain derived from its respective protein coding sequence during the amplification process. In one embodiment, this is accomplished through use of a proof-reading enzyme during amplification.
  • a proof-reading enzyme such as the bacteriophage phi 29 enzyme has been described.
  • suitable enzymes may include PhusionTM polymerase from NEB or (ultra)pfu enzymes from Stratagene.
  • kits including, e.g., Advantage® cDNA PCR Kits [Clontech, an enzyme blend consisting of an N-terminal deletion mutant of Taq DNA Polymerase, a proofreading enzyme, and TaqStart® Antibody],
  • PCR primer a Laboratory Manual, 2d ed., ed., Carl W. Dieffenbach, Gabriela S.
  • the method of the invention may be used to generate a combinatorial library containing altered protein coding sequences.
  • the resulting library of altered protein coding sequences can be used to general a combinatorial library of the proteins encoded by the altered coding sequences using expression methods known to those of skill in the art.
  • the library of altered protein coding sequences is used to general a viral library.
  • the viral capsid and/or envelope may be the product of the altered protein coding sequences.
  • another viral element may be altered protein coding sequence or the product thereof. Suitable techniques for generation of these protein and/or viral libraries from the combinatorial library prepared by the method of the invention are known to those of skill in the art.
  • an AAV capsid library may be generated in two additional steps [I. Virella-Lowell, et l, J Gene Med l: 842-850], e.g., by partially packaging the library containing recombinant Cap genes into AAV2 capsids by cotransfection of a suitable plasmid along with the AAV plasmid library and infecting the resulting AAV library into suitable host cells ⁇ e.g., HE 293 cells), followed by superinfection with adenovirus (Ad) dl309, thus ensuring production of chimeric AAV capsids packaging the corresponding chimeric AAV genome.
  • suitable host cells ⁇ e.g., HE 293 cells
  • Ad adenovirus
  • such an altered protein coding sequence is a protein coding sequence in which the original sequences of the ROI are conserved (unchanged), but in which ROI from different protein coding sequence sources are combined.
  • a library comprising an adeno-associated virus (AAV) capsid coding sequence made be generated through the method of the invention, involving the use of two or more different AAV capsid proteins.
  • AAV adeno-associated virus
  • such a library may be generated which retains the conserved regions of the capsid sequences of at least a first AAV and at least a second, different, AAV, such that the library contains various combinations of the hypervariable regions of each of the first and second AAVs superimposed on the unchanged conserved regions of the first and second AAVs.
  • At least one of the subdomains contains a first ROI and at least one of the subdomains contains a second ROI.
  • Primers are designed to specifically amplify these subdomains in a manner which permits the subdomains following treatment as described herein to be directionally and positionally assembled into a combinatorial library of the full-length protein coding sequence which contains altered sequences.
  • Each of the subdomains is amplified with at least one primer set which consists of a right primer and a left primer.
  • the primers are designed so that they are complimentary for the junctions between subdomains of the protein coding sequence which are contiguous with one another (both at the 5' and 3' end of each subdomain).
  • a given primer set is 5' identical to the subdomain located 5' to a selected subdomain and 3' identical to the subdomain located 3' to this selected subdomain.
  • the primers are independently selected from a length of about 1 5 to about 45 base pairs, about 20 to about 40 base pairs, about 25 to about 35 base pairs, or 30 base pairs.
  • a primer has complementarity at the 5' end of its subdomain template of 100% complementary over at least 8 base pairs.
  • a primer has 100% complementarity at the 3' end of its template (subdomain) over at least 15 base pairs.
  • Predetermined subsets of the amplified subdomains are subject to different amplification conditions which specifically generate unperturbed subdomains or which specifically generate altered subdomains.
  • the ROIs of interest are preselected to be treated under different conditions prior to pooling for assembling into a full-length altered protein coding sequence.
  • one or more ROIs may be preselected to have diversity introduced by altering their nucleotide sequences and one or more different ROIs may be preselected to remain conserved (i.e., no nucleotide sequences altered).
  • each may be independently modified using different techniques.
  • the method of the invention permits multiple ROIs to be treated in parallel by different treatments (e.g., introduce diversity or retain sequences without changes).
  • the amplified altered or unperturbed subdomains are pooled and assembled in a directional and positional manner. Typically this is achieved using splicing-by-overlap-extension (SOE) PCR [Horton, R.M., et al, Gene 77, 61-68 (1989)] to generate a combinatorial library which comprise the chimeric/hybrid and/or altered sequences.
  • SOE splicing-by-overlap-extension
  • the amplified subdomains termed alternatively herein building blocks, are assembled in a directional manner through the use of the 3 '-5 ' exonuclease activity of poxvirus DNA polymerase.
  • This enzyme is available commercially in kits, including, e.g., In-FusionTM cloning kit [Clontech ; Choo-ChooTM kit; ClonEZ® PCR cloning kit [GenScripfJ, FAST SEAMLESS PCRTM Cloning Kit [DoGene] .
  • In-FusionTM cloning kit [Clontech ; Choo-ChooTM kit; ClonEZ® PCR cloning kit [GenScripfJ, FAST SEAMLESS PCRTM Cloning Kit [DoGene] .
  • this method works by incubating linear duplex DNAs with homologous ends in the presence of Mg 2+ and low concentrations of dNTP, the 3'-5 ' proofreading activity of poxvirus DNA polymerase progressively removing nucleotides from the V end. This exposes complementary regions on the substrate DNAs that can spontaneously anneal through base pairing, resulting in seamless fusions.
  • the amplified subdomains will be assembled in the proper order and direction.
  • one of skill in the art may select another technique which permits directional and positional assembly of the subdomains to form a full-length, altered, protein coding sequence.
  • the SOE oligonucleotide PCR primers are the primers of the extreme 3' and 3' ends of the final assembled altered protein coding sequence. Such primers may be of the size described above for PCR primers and readily determined by one of skill in the art.
  • a two step method for generating diversity within delineated domains of protein coding sequence is provided.
  • two regions of interest (ROIs) are defined within a CDS.
  • the ROI are delineated by primers according to the method in a manner that all eventual permutations of the ROI universally link up with the flanking regions (FRs).
  • Diversity is generated by any number of methods in any number of combinations, e.g., one ROI be amplified from different templates without or without different primers or pools of primers whereas another ROI undergoes a DNA polymerization or ligation step that introduces variation through shuffling or mutagenesis.
  • the oligonucleotide primers are used to amplify the subdomain containing the region of interest in a manner that diversity is introduced through the use of different combinations of primers, through varying or providing a mixture of the template and/or modifying the PCR conditions (e.g. in shuffling or error- prone conditions).
  • e ft and P r i g h t are used in separate reactions and in combination with primers further outside (P ou tsideieft and Poutsideright, respectively) the region of interest to amplify the subdomains containing the flanking regions (Fig 1).
  • Flanking regions are combined with regions of interest in a final PCR reaction with all the internal components that one intends to combine in a combinatorial fashion as well as P ou «ideieft and Pomsideright- Subsequently, the entire protein coding sequence is amplified by SOE PC or another cloning process that then splices all individual components in a correct order due to the complementarity of the design of primers used in the first steps to amplify the subdomains. These full length libraries can then be screened and selected in a functional assay in order to identify hybrids and/or mutants with desirable properties.
  • Prior art methods are not as refined as the above described method in that they generate random diversity throughout a coding sequence.
  • the method of the invention allows structural and functional knowledge about the coding sequence to play into the combinatorial approach.
  • the net effect of this refinement is that functional diversity is generated more efficiently. For example, when diversity is generated across a coding region with other methods, this diversity will be introduced irrespective of whether that domain is a conserved or a variable domain. Often, conserved domain mutants are a priori defective.
  • the diversification can be focused on the variable domain. In addition, this can be done for multiple cis domains in a simultaneous manner to increase the combinatorial complexity that may allow for additive or synergistic functions to emerge or compensatory modifications to be incorporated.
  • the method of the invention permits one to control undesired sequence modifications within a selected subdomain (e.g., a constant region), permits reassembly of the various subdomains in the proper direction such that a full-length open reading frame is obtained, and permits control of subdomains such that a selected subdomain ⁇ e.g., a constant region or a hypervariable domain) is not.
  • a library of AAV capsid proteins e.g., vpl or a full-length capsid, vp2, or vp3
  • AAV particles may be desirable.
  • a library of AAV rep proteins may be desired.
  • the method of the invention permits one to generate and select novel, tailor-made AAV gene transfer vectors with desirable properties for clinical or commercial use.
  • the method of the invention is useful for generating combinatorial diversity in the hypervariable and/ or conserved regions of adeno- associated virus protein, a vector for gene therapy in order to select and identify novel vector with desirable properties.
  • AAV capsids by cryo- electron microscopy and/or X-ray crystallography.
  • AAV capsid functional variation such as receptor recognition (variable regions V, VIII, and IX), transduction efficiency (variable regions I, II-IV, V, VI, and IX), and antigenic recognition (variable regions I, III, IV, V, VI, VII, VIII, IX).
  • an ROI either to be changed or not changed within a selected AAV.
  • variable or constant regions which may be desirable ROIs by aligning the sequence of the AAVs and taking into consideration the information available on the three-dimensional structures of other AAV proteins.
  • Similar methods can be applied to other proteins and their coding sequences by taking into consideration the secondary and/or tertiary structure of the protein, a target domain (e.g., a binding site or epitope), and/or another functional domain.
  • a target domain e.g., a binding site or epitope
  • Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Examples of such programs include, “Clustal W”, “CAP Sequence Assembly”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using FastaTM, a program in GCG Version 6.1. FastaTM provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences.
  • FastaTM provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences.
  • percent sequence identity between nucleic acid sequences can be determined using FastaTM with its default parameters (a word size of 6 and the MOP AM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.
  • Multiple sequence alignment programs are also available for amino acid sequences, e.g., the "Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed.
  • one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).
  • HVR hypervariable regions
  • the HVR are located as follows: HVR1, aa 146-152; HVR2, aa 182-186; HVR3, aa 262-264; HVR4, aa 381-383; HVR5, aa 450-474; HVR6, aa 490-495; HVR7, aa 500-504; HVR8, aa 514- 522; HVR9, aa 534-555; HVR10, aa 581-594; HVR1 1, aa 658-667; and HVR12, aa 705-719 [the numbering system is based on an alignment which uses the AAV2 vpl as a point of reference].
  • Example 1 Engineering of Functional Chimera of AAV8 and rh32.33
  • AAV8 and rh32.33 are structurally and phylogenetically two of the most distinct primate AAVs. See, e.g., the dendogram of Fig. 2A.
  • Fig. 2B provides various quantitative measures of AAV8 and rh32.33 neutralization by human serum including intravenous immune globulin (IV1G) neutralization in the in vitro neutralizing antibody (NAB) assay (titer represents dilution of 50% transduction inhibition), estimate of seroprevalence in 888 individuals in worldwide populations (Europe, US, Africa and Australia) and lowest IVIG dose at which statistically significant in vivo transduction inhibition is observed at a dose of 10 M genome copies (GC) per mouse.
  • IV1G intravenous immune globulin
  • NAB in vitro neutralizing antibody
  • Fig. 3A is a cartoon showing the viral genome backbones for the pAAV and the pAAVivo. Whereas both systems receive BgllT-Spel flanked AAV capsids (here AAV8), pAAV yields replication-competent particles due to the presence of Rep in its wild typelike backbone.
  • CMV driven cap expression permits in vivo applications of the directed evolution system by rescue of genomes on an RNA level.
  • a first cycle PCR makes use of primers designed by a bioinformatics algorithm, and amplifies the hypervariable (H) and constant (C) domains on the AAV capsid structure.
  • H hypervariable
  • C constant
  • the primers are named using the following convention (X.Y#Pd), where X.Y defines the transition at the junction with X being name of the protein of the left fragment (5' on dsDNA) and Y the right fragment (3' on dsDNA); P for position defines the position of the junction on the defined domain i.e.
  • d for direction defines the direction of the primer, i.e., sense (s) or antisense (as).
  • sense i.e., sense
  • antisense i.e., antisense
  • 32.8IIEas is the antisense primer (as) that is located at the end (E) of hypervariable domain II (II) and defines a junction of rh32.33 (5') and AAV 8 (3') (32.8) of the final dsDNA molecule.
  • Ggtgaggttattggcgaccgtagtctc 32.81IEas 16 ccccagtacggctactgtggcattgtgact 8.32IIISS 17 cctcaatatggctacctaacactcaacaac 32.8IIISS 18 agtcacaatgccacagtagccgtactgggg 8.32IIISas 19
  • Atttcttccattcagggtatagtgggtgtc 32.8VEas 40 atcgctatggcaacagctggaccttcagat 8.32VI+VI1SS 41 cctccaatggcaacacacacaaagacgacgag 32.8VI+VIISS 42 atctgaaggtccagctgttgccatagcgat 8.32Vl+VllSas 43
  • Acttgtagatttgtaatagtttgaagtaa 32.8IXSas 60 aacaaccacctctacaagcaaatctccaac 8.8ISs 61 acctacttcggctacagcaccccctggggg 8.81Es 62
  • Tacacctccaactactacaaatctacaagt 8.8IXSs 75 gttaatacagaaggcgtgtactctgaaccc 8.8IXEs 76 aacaaccacttgtacctgcggctcggaaca 32.321Ss 77 acctacaacggattctccaccccctgggga 32.321ES 78
  • Ggtgaggttattggcgatggtcttggt 8.8IIEas 96 gttgttgagtgttaggtagccgtactgggggg 8.8IIISas 97 tccaggcagtagaaggaggagcgtcccac 8.81IIEas 98 ccgagacaagtagtacaggtactggtcaat 8.81VSas 99
  • the SOE oligonucleotide PCR primers in the first reaction are mixtures of primers with partial complimentarity to both AAV8 and AAVrh32.33 in their 5 ! and 3' extremities (Fig. 3C).
  • these 16 fragments ranging in sizes from -50 to 800bp, are amplified from AAV8 and rh32.33 and purified by electrophoresis.
  • final combinatorial domain diversification is accomplished by adding those fragments in a single reaction that aims at stitching them together in a directed but combinatorial fashion.
  • Fig. 3D final combinatorial domain diversification is accomplished by adding those fragments in a single reaction that aims at stitching them together in a directed but combinatorial fashion.
  • the resulting hybrid ORF library can be used to generate a library of AAVs with the hybrid capsids. This may be accomplished by cloning the hybrid ORFs into a suitable plasmid backbone pAAVivo (e.g., by cloning Spel into) and generating vectors using conventional methods.
  • the orfs can be cloned into the Bglll-Spel site of pAAVivo and subject to in vivo selection in an IVIG-passive transfer C57B16 mouse model. It is anticipated that capsids rescued by RT-PCR in this system will have undergone purifying selection for both liver transduction as well as lack of IVIG neutralization.
  • a diversified library of AAV8 capsids was prepared by diversifying the hypervariable region 8 (HVR8) [amino acids 583, 588, 594 - 597, based upon the numbering of the AAV 8 vpl, SEQ ID NO: 165].
  • HVR8 hypervariable region 8
  • the resulting capsids were assembled into vectors and assessed for their ability to be recognized by neutralizing antibodies (NABs). Those which avoided NABs were marked for further study.
  • a collection of AAV8 nucleic acid sequences within the open reading frame for hypervariable region 8 were amplified degenerate PCR primers.
  • Degenerate PCR primers are well suited for introducing controlled variation in this HPV (region of interest).
  • the goal is to generate AAV8 capsids having variants in the AAV8 HPV8 region in order to minimize or avoid the effect of neutralizing antibodies.
  • the PCR was performed at: 98° 10s, 66° 15s, 30 cycles.
  • the PCR was performed at 98° 10s 72°
  • a fragment of ⁇ 2.2kb was extracted with the electrophoresis of the PCR product, digested with Aarl (Fermentas) and Spel (NEW ENGLAND BIOLABS), and purified.
  • the purified fragment was ligated overnight by T4 DNA ligase at 16°C, with a 5478-bp fragment made by double digestion of pAAVivo plasmid, dephosphorylated and then purified.
  • the ligation was transformed into Escherichia coli Stbl4 (Invitrogen).
  • the transformation was transferred to TB medium (with 60 ⁇ g mL of carbenicillin), cultured overnight and the plasmid was extracted with EndoFree Plasmid Purification Mega Kit (Qiagen), This was the final plasmid for the mutagenesis library for AAV8 VP1 (amino acid 583, 588, 589, 594, 595, 596, and 597, VPl numbering, SEQ ID NO: 165).
  • helper plasmid AF6 1 .66 another helper plasmid (pRep) 0.83 ⁇ g and CaCl 2 (2.5M) 8.33 ⁇ .
  • the final volume was 83.3uL by adding water.
  • the mixture was then quickly mixed with an equal volume of 2x HEPES-buffered saline (HBS), and the total mixture was applied to 293 cells.
  • HBS 2x HEPES-buffered saline
  • the cells were harvested and lysated by 3 times of freeze/thaw, spun down at 13,000 rpm, 1 Ornin at room temperature; Benzonase(Merck) was then added to the supernatant (final concentration: 41.7 U/mL) and incubated at 37°C for 30 min.
  • the final product of AAV library was stored at -20°C.
  • Sequences in the Table below are listed in the order of homology to AAV8, with cDNA.9 being the most similar and cDNA.6 being the least similar. Based on the primary alignment, the sequences cluster in 4 groups (Gl: cDNA.9, gDNA.5 and gDNA.7; G2: cDNA.2, gDNA.2 and cDNA.3; G3: cDNA.8, cDNA.10 and gDNA.3; G4: gD A.l O, cD A.I and cDNA.6). * The mutation in this clone is outside the targeted area therefore testing for this clone was terminated.
  • GC genome copies
  • ADK.8 AAV8 Nab: 1 :2560
  • ADK.8 AAV8 Nab: 1 :2560
  • the cell culture was split at a ratio of 1 :5.
  • the cells were transfected with the plasmid AF6 and pRep (for 1 well of a 6-well plate, 2.49 of AF6 and 0.83 g of pRep).
  • the cells were harvested; RNA and genomic DNA were extracted from the cell.
  • RNA was converted to cDNA with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) with the random primer provided by the kit.
  • the sequences of the target region of AAV were retrieved by the following PCR.
  • the PCR conditions were as follows: 94° 30s, 61° 30s 72° 45 40cycles.
  • the PCR product was purified with QIAquickTM Gel Extraction
  • the resulting plasmids were treated with BsmBI and the 428-bp fragments were isolated and inserted back to the pAAV2/8 backbone using techniques known to one of ordinary skill in the art.
  • the pAAV2/8 plasmids carrying the inserts were submitted for sequencing and then used as the trans-plasmid in the following transfection to package individual AAV vector.
  • the packaging of the individual AAV vector was essentially as described in A above, with the pAAV2/8 plasmids carrying the inserts replacing the library plasmid and pAAV.CMV.EGFP replacing the pRep plasmid.

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Abstract

L'invention concerne un procédé pour la production dirigée d'une bibliothèque combinatoire d'une séquence codant pour une protéine modifiée. Ledit procédé consiste à : prédéterminer au moins deux fragments (régions) de séquence nucléotidique différents d'intérêt (ROI), dans lesquels la diversité de séquence doit être introduite de manière qui peut être différente pour les différents domaines. La séquence de codage de protéine pleine longueur est divisée en sous-domaines prédéterminés qui sont des fragments couvrant la séquence de codage de protéine pleine longueur. Au moins l'un des sous-domaines contient le premier ROI et au moins l'un des sous-domaines contient le second ROI. Des amorces sont conçues pour amplifier spécifiquement ces sous-domaines d'une manière qui permet aux sous-domaines suivant le traitement précité d'être assemblés en direction et en position en une bibliothèque combinatoire de la séquence de codage de protéine pleine longueur qui contient des séquences modifiées. Chacun des sous-domaines est amplifié au moyen d'une série d'ensembles d'amorces qui comprennent au moins un premier ensemble d'amorces et au moins un second ensemble d'amorces, chacun des ensembles d'amorces étant constitué d'une amorce droite et d'une amorce gauche complémentaires d'une jonction située entre des fragments consécutifs de la séquence de codage de protéine. Un sous-ensemble prédéterminé des sous-domaines amplifiés est soumis à des conditions d'amplification différentes qui génèrent des sous-domaines à séquences modifiées. Puis, les sous-domaines amplifiés sont regroupés et assemblés en direction et en position au moyen de SOE PCR ou d'autres procédés de clonage pour générer une bibliothèque combinatoire de séquences de codage de protéine pleine longueur qui comprennent les séquences modifiées.
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