WO2003023032A2 - High throughput directed evolution by rational mutagenesis - Google Patents

High throughput directed evolution by rational mutagenesis Download PDF

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WO2003023032A2
WO2003023032A2 PCT/IB2002/003921 IB0203921W WO03023032A2 WO 2003023032 A2 WO2003023032 A2 WO 2003023032A2 IB 0203921 W IB0203921 W IB 0203921W WO 03023032 A2 WO03023032 A2 WO 03023032A2
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seq
gcc
gcg
nucleic acid
protein
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WO2003023032A3 (en
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Manuel Vega
Lila Drittanti
Marjorie Flaux
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Nautilus Biotech
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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
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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
    • C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
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    • 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
    • C12N15/1075—Isolating an individual clone by screening libraries by coupling phenotype to genotype, not provided for in other groups of this subclass
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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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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    • 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
    • C12N2750/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • proteins and peptides include, but are not limited to, intracellular proteins, messenger/signaling/hormone proteins and viral proteins.
  • Directed evolution refers to biotechnological processes for optimizing the activity of proteins by means of random changes introduced into selected respective genes. Directed evolution involves the creation of a library of mutated genes, and then selection of the mutants that encode proteins having desired properties. The process can be an iterative one in which gene products that have improvement in a desired property are subjected to further cycles of mutation and screening. Directed evolution provides a way to adapt natural proteins to work in new chemical or biological environments, and/or to elicit new functions.
  • the .potential plasticity of proteins is such that chances exist that for every new challenge, such as a new environment and desired new or altered activity, it should be possible, given a sufficient pool of modified proteins (or encoding nucleic acids), that an appropriately 'evolved' protein could be found that would have a desired activity.
  • the problem is in generating and then identifying the appropriate sequence.
  • the methods provided herein include the steps of identifying a protein target of interest; obtaining nucleic acids that encode the target, which may be from any source, such as a natural library, a collection generated by known gene shuffling techniques and related methods, and, then creating variants of the proteins using methods for rational mutagenesis provided herein. Whatever method is used to select or generate the nucleic acids encoding the protein targets, each molecule is processed and screened separately in a high throughput format.
  • each nucleic acid molecule is introduced into an expression vector for expression in a bacterial cell or into a vector for expression in a eukaryotic host cell.
  • the nucleic acids of interest are introduced into host cells in an expression vector, such as by transfection for bacterial hosts and transduction with viral vectors into eukaryotic hosts with viral vectors.
  • Each variant is introduced into a host and the resulting cells are maintained separately, such as in an addressable array of wells in a microtiter plate or other substrate with discrete locations for performing reactions or retaining molecules of interest.
  • Typical formats are 96 loci, and multiples thereof (384, 1536, 3072, . . . 96 x n, where n is 1 to any number desired, such as 10, 20, 30, 50 . . .100), although any convenient number of loci may be employed.
  • the process is conducted in a high throughput format, for many embodiments, it is often important to assess the relative numbers of transformed, transduced or transfected cells.
  • the relative (or actual) titer of the vector such as the recombinant viral vector, must be known to permit analysis of results.
  • the processes require accurate titering of the viruses in a collection or among collections (libraries) so that the activities of the screened mutant proteins can be compared.
  • nucleic acid molecules encoding variants are prepared. Each variant encoded by the nucleic acid molecules has a single amino acid replaced with another selected amino acid, such as alanine (Ala), glycine (Gly), serine (Ser) or any other suitable amino acid, typically one selected to have a neutral effect on secondary and tertiary structure.
  • Ala alanine
  • Gly glycine
  • Ser serine
  • the resulting series of variants are separately screened in the high throughput format provided herein, and those that have a change in the target activity are selected and the modified amino acids are designated "hits.”
  • Nucleic acid molecules encoding proteins in which each hit position is replaced by the eighteen remaining amino acids then are synthesized and the resulting collection of molecules is screened, such by introduction into host cells, and the proteins that result in an improvement of a targeted activity, are identified. Such proteins are designated “leads.”
  • Leads may be further modified by producing proteins that have combinations of the mutations identified in the leads. This method, which does not require any knowledge of the structure of a target protein, permits precise control of locations where changes are introduced and also the amount of change that is introduced.
  • the high throughput directed evolution processes provided herein include the use of virus libraries containing mutant versions of a gene; viral libraries of such mutant genes are also provided.
  • Reporter cells are infected with the titered viruses that encode the mutant genes.
  • the mutant genes are expressed and read-out data from either biochemical or cell-based assays, while isolating each mutant/virus physically from the others (i.e. one-by-one analysis), are collected and analyzed.
  • Serial dilution assays i.e. a series of dilutions for each individual mutant/virus in the library
  • the biochemical/cell- based assays are performed on each single dilution for each individual mutant/virus.
  • Analysis of the serial dilution readout-data can be performed using any method of analysis that permits one-by-one comparisons. Hill-based analysis (see, published International PCT application No.
  • WO 01 /44809 based on PCT n ° PCT/FR00/03503, Dec, 2000, and the description herein) are employed for analysis of the data. Protein/protein domain variants identified using the methods are also provided. Also provided are nucleic acid molecules and proteins and polypeptides produced by the methods and viruses and cells that contain the nucleic acid molecules and proteins.
  • the process of rational directed evolution provided herein is applied to the AAV rep gene.
  • the resulting recombinant rep protein variants and rAAV are also provided.
  • the rep proteins are those that result in increased rAAV production in rAAV that encode such mutants, thereby, among a variety of advantages, offering a solution to the need in the gene therapy industry to increase the production therapeutic vectors without up-scaling manufacturing.
  • some methods provided herein have been used to identify amino acid "hit" positions in adeno-associated virus (AAV) rep proteins that are relevant for AAV or rAAV production.
  • AAV adeno-associated virus
  • Those amino acid positions are such that a change in the amino acid leads to a change in protein activity either to lower activity or to higher activity compared to native-sequence Rep proteins.
  • the hit positions were then used to generate further mutants designated "leads.”
  • the resulting mutant rep proteins that result in either higher or lower levels of AAV or rAAV virus compared to the wild-type (native) Rep protein(s).
  • the rep mutants are those that inhibit papillomavirus (PV) and PV-associated diseases, including certain cancers and human immunodeficiency virus (HIV) and HIV-associated diseases.
  • FIGURES 1 A - 1 E summarize various exemplary embodiments of the high throughput processes provided herein.
  • FIGURE 1 A depicts an embodiment of the process in which an amino acid scan is employed to generate a library of mutants, which are then introduced into viral vectors, such as an adeno-associated viral vector (AAV), a herpes virus, such as herpes simplex virus (HSV) and other herpes virus vectors, a vaccinia virus vector, retroviral vectors, such as MuMLV, MoMLV, feline leukemia virus, and HIV and other lentiviruses, adenovirus vectors and other suitable viral vector, each member of the library is individually tested and phenotypically characterized to identify HITS.
  • AAV adeno-associated viral vector
  • HSV herpes simplex virus
  • retroviral vectors such as MuMLV, MoMLV, feline leukemia virus, and HIV and other lentiviruses
  • adenovirus vectors and other suitable viral vector
  • FIGURE 1 B summarizes round 2 in which LEADS are developed by mutagenesis at and/or surrounding the positions identified as HITS;
  • FIGURE 1 C summarizes the optional next round in which recombination among LEADS is performed to further optimize the LEADS;
  • FIGURE 1 D depicts the process in mammalian cells; and
  • FIGURE 1 E depicts the process in bacterial cells.
  • FIGURE 2A depicts an exemplary titering process (in this instance the TREE " for titering AAV) in a 96 well format;
  • FIGURE 2B shows the results and analysis of a titering process performed using the TREE TM procedure; and
  • FIGURE 2C shows an exemplary calibration curve for the calculation of the titer using the TREE TM method.
  • FIGURE 3A and 3B depict "HITS” and “LEADS” respectively for identification of AAV rep mutants "evolved” for increased activity.
  • FIGURE 4 shows the genetic map of AAV, including the location of promoters, and transcripts; amino acid 1 of the Rep 78 gene is at nucleotide 321 in the AAV-2 genome.
  • FIGURES 5A and 5B show the alignment of amino acid sequences of Rep78 among AAV-1 ; AAV-6; AAV-3; AAV-3B; AAV-4; AAV-2; AAV- 5 sequences, respectively; the hit positions with 100 percent homology among the serotypes are bolded italics, where the position is different (compared to AAV-2, no. 6 in the Figure) in a particular serotype, it is in bold; a sequence indicating relative conservation of sequences among the serotypes is labeled "C".
  • directed evolution refers to methods that adapt natural proteins or protein domains to work in new chemical or biological environments and/or to elicit new functions. It is more a more broad- based technology than DNA shuffling.
  • high-throughput screening refers to processes that test a large number of samples, such as samples of test proteins or cells containing nucleic acids encoding the proteins of interest to identify structures of interest or to identify test compounds that interact with the variant proteins or cells containing them. HTS operations are amenable to automation and are typically computerized to handle sample preparation, assay procedures and the subsequent processing of large volumes of data.
  • DNA shuffling is a PCR-based technology that produces random rearrangements between two or more sequence-related genes to generate related, although different, variants of given gene.
  • hits are mutant proteins that have an alteration in any attribute, chemical, physical or biological property in which such alteration is sought.
  • hits are generally generated by systematically replacing each amino acid in a protein or a domain thereof with a selected amino acid, typically Alanine, Glycine, Serine or any amino acid, as long as each residue is replaced with the same residue.
  • Hits may be generated by other methods known to those of skill in the art and tested by the high throughput methods herein.
  • a Hit typically has activity with respect to the function of interest that differs by at least 10%, 20%, 30% or more from the wild type or native protein.
  • the desired alteration which is generally a reduction in activity, will depend upon the function or property of interest.
  • leads are “hits” whose activity has been optimized for the particular attribute, chemical, physical or biological property.
  • leads are generally produced by systematically replacing the hit loci with all remaining 18 amino acids, and identifying those among the resulting proteins that have a desired activity. The leads may be further optimized by replacement of a plurality of "hit” residues. Leads may be generated by other methods known to those of skill in the art and tested by the high throughput methods herein.
  • a lead typically has activity with respect to the function of interest that differs from the native activity, by a desired amount and is at by at least 10%, 20%, 30% or more from the wild type or native protein.
  • a Lead will have an activity that is 2 to 10 or more times the native protein for the activity of interest. As with hits, the change in the activity is dependent upon the activity that is "evolved.” Th e desired alteration will depend upon the function or property of interest.
  • MOI multiplicity of infection
  • ip with reference to a virus or recombinant vector, refers to a titer of infectious particles.
  • pp refers to the total number of vector (or virus) physical particles
  • biological and pharmacological activity includes any activity of a biological pharmaceutical agent and includes, but is not limited to, biological efficiency, transduction efficiency, gene/transgene expression, differential gene expression and induction activity, titer, progeny productivity, toxicity, cytotoxicity, immunogenicity, cell proliferation and/or differentiation activity, anti-viral activity, morphogenetic activity, teratogenetic activity, pathogenetic activity, therapeutic activity, tumor suppressor activity, ontogenetic activity, oncogenetic activity, enzymatic activity, pharmacological activity, cell/tissue tropism and delivery.
  • output signal refers to parameters that can be followed over time and, if desired, quantified. For example, when a virus infects a cell, the infected cell undergoes a number of changes. Any such change that can be monitored and used to assess infection, is an "output signal," and the cell is referred to as a "reporter cell.” Output signals include, but are not limited to, enzyme activity, fluorescence, luminescence, amount of product produced and other such signals. Output signals include expression of a viral gene or viral gene product, including heterologous genes (transgenes) inserted into the virus.
  • transgenes heterologous genes
  • Such expression is a function of time (“t") after infection, which in turn is related to the amount of virus used to infect the cell, and, hence, the concentration of virus ("s") in the infecting composition.
  • t time
  • s concentration of virus
  • the output signal is higher.
  • the output signal increases as a function of time until a plateau is reached.
  • Output signals may also measure the interaction between cells, expressing heterologous genes, and biological agents.
  • adeno-associated virus AAV is a defective and non-pathogenic parvovirus that requires co-infection with either adenovirus or herpes virus for its growth and multiplication, able of providing helper functions.
  • a variety of serotypes are known, and contemplated herein.
  • Such serotypes include, but are not limited to: AAV-1 (Genbank accession no. NC002077; accession no. VR-645); AAV- 2 (Genbank accession no. NC001401 ; accession no. VR-680); AAV-3 (Genbank accession no. NC001 729; accession no. VR-681 ); AAV-3b (Genbank accession no. NC001863); AAV-4 (Genbank accession no. NC001 829; ATCC accession no. VR-646 ); AAV-6 (Genbank accession no.NC001862); and avian associated adeno-virus (ATCC accession no. VR-1449) .
  • AAVs as vectors for gene expression in vitro and for in vivo use for gene therapy are well known (see, e.g. , U.S. Patent Nos. 4,797,368, 5, 1 39,941 , 5,798,390 and 6, 1 27, 1 75; Tessier et al. (2001 ) . Virol. 75:375-383; Salvetti et al. (1 998) Hum Gene Ther 20:695-706; Chadeuf et al. (2000) J Gene Med 2:260-268) .
  • the activity of a Rep protein or of a capsid protein refers to any biological activity that can be assessed.
  • the activity assessed for the rep proteins is the amount [i.e. , titer) of AAV produced by a cell.
  • y is the variable being measured, such as a response, signal
  • y max is the maximal response achievable
  • [D] is the molar concentration of a drug
  • [D 50 ] is the concentration that produces a 50% maximal response to the drug
  • n is the slope parameter, which is 1 if the drug binds to a single site and with no cooperativity between or among sites.
  • a Hill plot is log 10 of the ratio of ligand-occupied receptor to free receptor vs. log [D] (M) .
  • the slope is n, where a slope of greater than 1 indicates cooperativity among binding sites, and a slope of less than 1 can indicate heterogeneity of binding.
  • This general equation has been employed for assessing interactions in complex biological systems (see, published International PCT application No. WO 01 /44809 based on PCT n ° PCT/FR00/03503, see, also, EXAMPLES).
  • K constant of resistance of the assay system to elicit a response to a biological agent
  • e global efficiency of the process or reaction triggered by the biological agent on the assay system
  • r is the apparent titer of the biological agent
  • ⁇ is the absolute titer of the biological agent
  • ⁇ is the heterogeneity of the biological process or reaction.
  • the parameters ⁇ (potency) or K (constant of resistance) are used to respectively assess the potency of a test agent to produce a response in an assay system and the resistance of the assay system to respond to the agent.
  • e(efficiency) is the slope at the inflection point of the Hill curve (or, in general, of any other sigmoidal or linear approximation), to assess the efficiency of the global reaction (the biological agent and the assay system taken together) to elicit the biological or pharmacological response.
  • ⁇ (apparent titer) is used to measure the limiting dilution or the apparent titer of the biological agent.
  • ⁇ absolute titer
  • ⁇ absolute titer
  • ⁇ (heterogeneity) measures the existence of discontinuous phases along the global reaction, which is reflected by an abrupt change in the value of the Hill coefficient or in the constant of resistance.
  • a library of mutants refers to a collection of plasmids or other vehicles that carry (encode) the gene variants, such that individual plasmids or other vehicles carry individual gene variants.
  • a "reporter cell” is the cell that "reports”, i.e., undergoes the change, in response to introduction of the nucleic acid infection and, therefore, it is named here a reporter cell.
  • reporter or “reporter moiety” refers to any moiety that allows for the detection of a molecule of interest, such as a protein expressed by a cell.
  • Typical reporter moieties include, for example, fluorescent proteins, such as red, blue and green fluorescent proteins.
  • nucleic acid encoding the reporter moiety can be expressed as a fusion protein with a protein of interest or under the control of a promoter of interest.
  • a titering virus increases or decreases the output signal from a reporter virus, which is a virus that can be detected, such as by a detectable label or signal.
  • phenotype refers to the physical or other manifestation of a genotype (a sequence of a gene). In the methods herein, phenotypes that result from alteration of a genotype are assessed.
  • activity refers to the function or property to be evolved.
  • An active site refers to a site(s) responsible or that participates in conferring the activity or function.
  • the activity or active site evolved (the function or property and the site conferring or participating in conferring the activity) may have nothing to do with natural activities of a protein. For example, it could be an 'active site' for conferring immunogenicity (immunogenic sites or epitopes) on a protein.
  • amino acids which occur in the various amino acid sequences appearing herein, are identified according to their known, three-letter or one-letter abbreviations (see, Table 1 ).
  • nucleotides which occur in the various nucleic acid fragments, are designated with the standard single-letter designations used routinely in the art.
  • amino acid residue refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages.
  • the amino acid residues described herein are presumed to be in the "L" isomeric form. Residues in the "D" isomeric form, which are so- designated, can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide.
  • amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus.
  • amino acid residue is broadly defined to include the amino acids listed in the Table of Correspondence and modified and unusual amino acids, such as those referred to in 37 C.F.R. ⁇ ⁇ 1 .821 - 1 .822, and incorporated herein by reference.
  • a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to an amino-terminal group such as NH 2 or to a carboxyl-terminal group such as COOH.
  • nucleic acids include DNA, RNA and analogs thereof, including protein nucleic acids (PNA) and mixture thereof.
  • PNA protein nucleic acids
  • Nucleic acids can be single or double stranded.
  • probes or primers optionally labeled, with a detectable label, such as a fluorescent or radiolabel
  • single-stranded molecules are contemplated. Such molecules are typically of a length such that they are statistically unique of low copy number (typically less than 5, preferably less than 3) for probing or priming a library.
  • a probe or primer contains at least 14, 1 6 or 30 contiguous of sequence complementary to or identical to a gene of interest.
  • Probes and primers can be 10, 1 4, 1 6, 20, 30, 50, 100 or more nucleic acid bases long.
  • homologous means about greater than 25% nucleic acid sequence identity, preferably 25% 40%, 60%, 80%, 90% or 95%.
  • sequences are aligned so that the highest order match is obtained (see, e.g..' Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1 988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
  • nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.
  • nucleic acid homolog refers to a nucleic acid that includes a preselected conserved nucleotide sequence, such as a sequence encoding a therapeutic polypeptide.
  • substantially homologous it is meant having at least 80%, preferably at least 90%, most preferably at least 95% homology therewith or a less percentage of homology or identity and conserved biological activity or function.
  • homology and “identity” are often used interchangeably. In this regard, percent homology or identity may be determined, for example, by comparing sequence information using a GAP computer program. The GAP program uses the alignment method of Needleman and Wunsch [J. Mol. Biol.
  • the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.
  • the preferred default parameters for the GAP program may include: (1 ) a unitary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.
  • nucleic acid molecules have nucleotide sequences that are, for example, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% /'identical” can be determined using known computer algorithms such as the "FAST A" program, using for example, the default parameters as in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 55:2444 ( 1 988) .
  • the BLAST function of the National Center for Biotechnology Information database may be used to determine identity
  • sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques.
  • identity is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 45: 1073 (1 988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1 994, and Carillo, H. & Lipton, D., SIAM J Applied Math 45: 1 073 (1 988).
  • Methods to determine identity and similarity are codified in computer programs.
  • Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 72(/):387 ( 1 984)), BLASTP, BLASTN, FASTA (Atschul, S.F., et a/., J Mo/ec Biol 2 /5:403 (1 990)), and CLUSTALW.
  • GCG program package Disevereux, J., et al., Nucleic Acids Research 72(/):387 ( 1 984)
  • BLASTP BLASTP
  • BLASTN BLASTN
  • FASTA Altschul, S.F., et a/., J Mo/ec Biol 2 /5:403 (1 990)
  • CLUSTALW for sequences displaying a relatively high degree of homology, alignment can be effected manually by simply lining up the
  • the term "identity" represents a comparison between a test and a reference polypeptide or polynucleotide.
  • a test polypeptide may be defined as any polypeptide that is 90% or more identical to a reference polypeptide.
  • CLUSTALW program was employed with parameters set as follows: scoring matrix BLOSUM, gap open 10, gap extend 0.1 , gap distance 40% and transitions/transversions 0.5; specific residue penalties for hydrophobic amino acids (DEGKNPQRS), distance between gaps for which the penalties are augmented was 8, and gaps of extremities penalized less than internal gaps.
  • a "corresponding" position on a protein refers to an amino acid position based upon alignment to maximize sequence identity.
  • AAV Rep proteins an alignment of the Rep 78 protein from AAV-2 and the corresponding protein from other AAV serotypes (AAV-1 , AAV-6, AAV-3, AAV-3B, AAV-4, AAV-2 and AAV-5) is shown in Figure 5.
  • the “hit” positions are shown in italics.
  • the term at least "90% identical to” refers to percent identities from 90 to 100% relative to the reference polypeptides.
  • Identity at a level of 90% or more is indicative of the fact that, ' assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared. No more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptides. Similar comparisons may be made between a test and reference polynucleotides. Such differences may be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they may be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10/100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, or deletions.
  • genetic therapy involves the transfer of heterologous nucleic acids to the certain cells, target cells, of a mammal, particularly a human, with a disorder or conditions for which such therapy is sought.
  • the nucleic acid, such as DNA is introduced into the selected target cells in a manner such that the heterologous nucleic acid, such as DNA, is expressed and a therapeutic product encoded thereby is produced.
  • the heterologous nucleic acid, such as DNA may in some manner mediate expression of DNA that encodes the therapeutic product, or it may encode a product, such as a peptide or RNA that in some manner mediates, directly or indirectly, expression of a therapeutic product.
  • Genetic therapy may also be used to deliver nucleic acid encoding a gene product that replaces a defective gene or supplements a gene product produced by the mammal or the cell in which it is introduced.
  • the introduced nucleic acid may encode a therapeutic compound, such as a growth factor or inhibitor thereof, or a tumor necrosis factor or inhibitor thereof, such as a receptor therefor, that is not normally produced in the mammalian host or that is not produced in therapeutically effective amounts or at a therapeutically useful time.
  • the heterologous nucleic acid, such as DNA, encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof.
  • Genetic therapy may also involve delivery of an inhibitor or repressor or other modulator of gene expression.
  • heterologous or foreign nucleic acid such as DNA and RNA
  • DNA and RNA are used interchangeably and refer to DNA or RNA that does not occur naturally as part of the genome in which it is present or which is found in a location or locations in the genome that differ from that in which it occurs in nature.
  • Heterologous nucleic acid is generally not endogenous to the cell into which it is introduced, but has been obtained from another cell or prepared synthetically. Generally, although not necessarily, such nucleic acid encodes RNA and proteins that are not normally produced by the cell in which it is expressed. Any DNA or RNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which it is expressed is herein encompassed by heterologous DNA.
  • Heterologous DNA and RNA may also encode RNA or proteins that mediate or alter expression of endogenous DNA by affecting transcription, translation, or other regulatable biochemical processes.
  • heterologous nucleic acid include, but are not limited to, nucleic acid that encodes traceable marker proteins, such as a protein that confers drug resistance, nucleic acid that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, and DNA that encodes other types of proteins, such as antibodies.
  • heterologous DNA or foreign DNA includes a DNA molecule not present in the exact orientation and position as the counterpart DNA molecule found in the genome. It may also refer to a DNA molecule from another organism or species (i.e. , exogenous) .
  • a therapeutically effective product introduced by genetic therapy is a product that is encoded by heterologous nucleic acid, typically DNA, that, upon introduction of the nucleic acid into a host, a product is expressed that ameliorates or eliminates the symptoms, manifestations of an inherited or acquired disease or that cures the disease.
  • isolated with reference to a nucleic acid molecule or polypeptide or other biomolecule means that the nucleic acid or polypeptide has separated from the genetic environment from which the polypeptide or nucleic acid were obtained. It may also mean altered from the natural state. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Thus, a polypeptide or polynucleotide produced and/or contained within a recombinant host cell is considered isolated.
  • isolated polypeptide or an “isolated polynucleotide” are polypeptides or polynucleotides that have been purified, partially or substantially, from a recombinant host cell or from a native source.
  • a recombinantly produced version of a compounds can be substantially purified by the one-step method described in Smith and Johnson, Gene 57/31 -40 (1 988). The terms isolated and purified are sometimes used interchangeably.
  • isolated it is meant that the nucleic is free of the coding sequences of those genes that, in the naturally-occurring genome of the organism (if any) immediately flank the gene encoding the nucleic acid of interest.
  • Isolated DNA may be single-stranded or double-stranded, and may be genomic DNA, cDNA, recombinant hybrid DNA, or synthetic DNA. It may be identical to a native DNA sequence, or may differ from such sequence by the deletion, addition, or substitution of one or more nucleotides.
  • Isolated or purified as it refers to preparations made from biological cells or hosts means any cell extract containing the indicated DNA or protein including a crude extract of the DNA or protein of interest.
  • a purified preparation can be obtained following an individual technique or a series of preparative or biochemical techniques and the DNA or protein of interest can be present at various degrees of purity in these preparations.
  • the procedures may include for example, but are not limited to, ammonium sulfate fractionation, gel filtration, ion exchange change chromatography, affinity chromatography, density gradient centrifugation and electrophoresis.
  • a preparation of DNA or protein that is "substantially pure” or “isolated” should be understood to mean a preparation free from naturally occurring materials with which such DNA or protein is normally associated in nature. "Essentially pure” should be understood to mean a “highly” purified preparation that contains at least 95% of the DNA or protein of interest.
  • a ceil extract that contains the DNA or protein of interest should be understood to mean a homogenate preparation or cell-free preparation obtained from cells that express the protein or contain the DNA of interest.
  • the term "cell extract” is intended to include culture media, especially spent culture media from which the cells have been removed.
  • receptor refers to a biologically active molecule that specifically binds to (or with) other molecules.
  • receptor protein may be used to more specifically indicate the proteinaceous nature of a specific receptor.
  • recombinant refers to any progeny formed as the result of genetic engineering.
  • a promoter region refers to the portion of DNA of a gene that controls transcription of the DNA to which it is operatively linked.
  • the promoter region includes specific sequences of DNA that are sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as the promoter.
  • the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of the RNA polymerase. These sequences may be cis acting or may be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, may be constitutive or regulated.
  • operatively linked generally means the sequences or segments have been covalently joined into one piece of DNA, whether in single or double stranded form, whereby control or regulatory sequences on one segment control or permit expression or replication or other such control of other segments.
  • the two segments are not necessarily contiguous.
  • a DNA sequence and a regulatory sequence(s) are connected in such a way to control or permit gene expression when the appropriate molecules, e.g., transcriptional activator proteins, are bound to the regulatory sequence(s).
  • production by recombinant means by using recombinant DNA methods means the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA, including cloning expression of genes and methods, such as gene shuffling and phage display with screening for desired specificities.
  • a splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that results in more than one type of mRNA.
  • composition refers to any mixture of two or more products or compounds. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.- As used herein, a combination refers to any association between two or more items.
  • substantially identical to a product means sufficiently similar so that the property of interest is sufficiently unchanged so that the substantially identical product can be used in place of the product.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
  • Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form, are not bound to the chromosome.
  • Plasmid and vector are used interchangeably as the plasmid is the most commonly used form of vector. Other such other forms of expression vectors that serve equivalent functions and that become known in the art can be used subsequently hereto. As used herein, vector is also used interchangeable with “virus vector” or “viral vector” . In this case, which will be clear from the context, the “vector” is not self-replicating. Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.
  • transduction refers to the process of gene transfer and expression into mammalian and other cells mediated by viruses.
  • Transfection refers to the process when mediated by plasmids.
  • polymorphism refers to the coexistence of more than one form of a gene or portion thereof.
  • a portion of a gene of which there are at least two different forms, i.e., two different nucleotide sequences, is referred to as a "polymorphic region of a gene".
  • a polymorphic region can be a single nucleotide, referred to as a single nucleotide polymorphism (SNP), the identity of which differs in different alleles.
  • SNP single nucleotide polymorphism
  • a polymorphic region can also be several nucleotides in length.
  • polymorphic gene refers to a gene having at least one polymorphic region.
  • allele which is used interchangeably herein with “allelic variant” refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides. An allele of a gene can also be a form of a gene containing a mutation.
  • gene refers to a nucleic acid molecule comprising an open reading frame and including at least one exon and (optionally) an intron sequence.
  • a gene can be either RNA or DNA. Genes may include regions preceding and following the coding region (leader and trailer) .
  • intron refers to a DNA sequence present in a given gene which is spliced out during mRNA maturation.
  • nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: x refers to the nucleotide sequence of the complementary strand of a nucleic acid strand having SEQ ID NO: x.
  • complementary strand is used herein interchangeably with the term “complement” .
  • the complement of a nucleic acid strand can be the complement of a coding strand or the complement of a non-coding strand.
  • the complement of a nucleic acid having SEQ ID NO: x refers to the complementary strand of the strand having SEQ ID NO: x or to any nucleic acid having the nucleotide sequence of the complementary strand of SEQ ID NO: x.
  • the complement of this nucleic acid is a nucleic acid having a nucleotide sequence which is complementary to that of SEQ ID NO: x.
  • the term "coding sequence" refers to that portion of a gene that encodes an amino acid sequence of a protein.
  • sense strand refers to that strand of a double-stranded nucleic acid molecule that has the sequence of the mRNA that encodes the amino acid sequence encoded by the double- stranded nucleic acid molecule.
  • an array refers to a collection of elements, such as nucleic acid molecules, containing three or more members.
  • An addressable array is one in which the members of the array are identifiable, typically by position on a solid phase support or by virtue of an identifiable or detectable label, such as by color, fluorescence, electronic signal (i.e. radiofrequency (RF), microwave or other frequency that does not substantially alter the interaction of the molecules of interest), bar code or other symbology, chemical or other such label.
  • RF radiofrequency
  • the members of the array are immobilized to discrete identifiable loci on the surface of a solid phase or directly or indirectly linked to or otherwise associated with the identifiable label, such as affixed to a microsphere or other particulate support (herein referred to as beads) and suspended in solution or spread out on a surface.
  • a microsphere or other particulate support herein referred to as beads
  • a support also referred to as a matrix support, a matrix, an insoluble support or solid support
  • a molecule of interest typically a biological molecule, organic molecule or biospecific ligand is linked or contacted.
  • Such materials include any materials that are used as affinity matrices or supports for chemical and biological molecule syntheses and analyses, such as, but are not limited to: polystyrene, polycarbonate, polypropylene, nylon, glass, dextran, chitin, sand, pumice, agarose, polysaccharides, dendrimers, buckyballs, polyacrylamide, silicon, rubber, and other materials used as supports for solid phase syntheses, affinity separations and purifications, hybridization reactions, immunoassays and other such applications.
  • the matrix herein can be particulate or can be in the form of a continuous surface, such as a microtiter dish or well, a glass slide, a silicon chip, a nitrocellulose sheet, nylon mesh, or other such materials.
  • the particles When particulate, typically the particles have at least one dimension in the 5-10 mm range or smaller.
  • Such particles referred collectively herein as "beads”, are often, but not necessarily, spherical. Such reference, however, does not constrain the geometry of the matrix, which may be any shape, including random shapes, needles, fibers, and elongated. Roughly spherical "beads", particularly microspheres that can be used in the liquid phase, are also contemplated.
  • the “beads” may include additional components, such as magnetic or paramagnetic particles (see, e.g.,, Dyna beads (Dynal, Oslo, Norway)) for separation using magnets, as long as the additional components do not interfere with the methods and analyses herein.
  • matrix or support particles refers to matrix materials that are in the form of discrete particles.
  • the particles have any shape and dimensions, but typically have at least one dimension that is 100 mm or less, 50 mm or less, 10 mm or less, 1 mm or less, 100 ⁇ m or less, 50 ⁇ m or less and typically have a size that is 100 mm 3 or less, 50 mm 3 or less, 10 mm 3 or less, and 1 mm 3 or less, 100 ⁇ m 3 or less and may be order of cubic microns.
  • Such particles are collectively called "beads.”
  • the abbreviations for any protective groups, amino acids and other compounds are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1 972) Biochem. 7 7:942-944).
  • the processes include methods that are particularly adapted for high throughput protocols, which require accurate methods for identifying modified proteins.
  • a general directed evolution process includes the following steps: 1 . Generation of diversity at the nucleic acid level, on the gene to be 'evolved'
  • the processes provided herein effect these steps that can be performed in a high throughput format (see, FIGURES 1 ) that is optionally automated.
  • a distinguishing characteristic of the processes provided herein is that each candidate nucleic acid molecule is separately generated and screened.
  • In an automated process at least some of the steps are performed without human intervention and are generally controlled by software.
  • Most, if not all steps, are performed in addressable formats, such as at discrete locations in or on solid supports, such as microtiter plates or in other addressable formats, such as linked to coded supports.
  • the supports can be electronically, physically, chemically or otherwise identifiable, such as by an identifiable symbology, including a bar code, or can be color coded.
  • a semi-rational approach to creating diversity or evolving genes is provided herein.
  • the goal is to create diversity but to decrease the number of molecules to be screened.
  • the molecules can be screened in high throughput format molecule-by- molecule (or groups thereof).
  • step 1 Generation of diversity at the nucleic acid level, in principle, can be accomplished by a number of diverse technologies like mutagenesis (either site-directed or random), recombination, shuffling and de-novo synthesis. These different technologies differ in the degree of diversity they generate as well as in the minimal length of the unitary change they can introduce (from single base to large domains).
  • the outcome of step 1 is a collection of diverse, although highly related, molecules that constitutes what is called a 'library' .
  • This step is crucial, since it provides the initial conditions for the entire process and is determinative of the outcome.
  • the chances of finding an optimized gene version in a library is a function of the total diversity present in the library.
  • the type of diversity introduced (such as, but not limited to, single point mutations, multiple point mutations, scarce small rearrangements, recombination of large domains, multiple shuffling) condition the outcome, particularly with respect to the generation of new variants compared to the original gene, and the probability that the new variants, not only exhibit the "evolved" function or property, but also work in their natural biological networks where they are expected to act by interacting, recognizing, and/or being recognized, by a large panoply of other proteins and other molecules. Rapid discovery of protein variants at the amino acid level by rational mutagenesis (aa-scan)
  • a method referred to herein as an amino-acid scan method for directed evolution, is provided herein for generating protein variants.
  • This method can be performed on an entire protein or selected domains thereof, or can be used to identify benchmark sequences, such as functional domains, and, for example, recombine them as exchangeable units or restrict the diversity to limited or specific regions of the protein.
  • this method can be used with the processes provided herein, but it also has applications for any methods that use such variants or require generation of such variants, such as, but not limited to, searches for consensus sequences and homology regions that are used in functional genomics, functional proteomics; comparative modeling in protein crystallography and protein modeling; searches for natural diversity, (e.g.
  • exon- or family-shuffling-based diversity e.g., directed evolution using gene shuffling (see, e.g., U.S
  • the amino-acid scanning-based method provided herein has advantages that prior methods do not have.
  • prior methods are based upon the underlying assumption that there are parts of the molecule (gene or protein) that are sufficiently adapted to perform their respective function, and further changes are not advantageous.
  • Such methods do not look at total potential plasticity of a given molecule, but at the plasticity still permitted while keeping some basic functions in place. By choosing this route, however, additional potential variation is missed.
  • the potential in the intrinsic plasticity of those regions that are presumed 'preserved' is lost. For instance, methods (e.g. , those in U.S. Patent Nos.
  • the method provided herein is based on the premise that there are single amino acids or small blocks of sequence of amino acids that are either (1 ) directly involved in the activities that the methods 'evolve' (these amino acids would be at or close to the 'active sites' of the protein); or (2) directly involved in maintaining within the protein the intramolecular environment that allows the active site(s) to stay active.
  • amino acid-scanning has been used to precisely identify those amino acids directly involved in the active sites of some enzymes and receptors (see, e.g., Becl-Sickinger et al. (1994) Eur. J. Biochem. 223:947-958; Gibbs et al. (1991 )J. Biol. Chem. 255:8923-8931 ; Matsushita et al. (2000) J. Biol. Chem. 275:l ⁇ OAA- 1 1049) but has not been employed for directed evolution or for the generation of diversity.
  • the amino acid scan as practiced in the prior art is used to produce a set of protein mutants, often within the region suspected to contain the active site(s), such that in each individual mutant a selected residue, such as Ala, replaces a different amino acid.
  • Ala or other neutral amino acids generally, although not necessarily, is selected as a replacement amino acid since, except in instances in which the replaced amino acid is directly involved in an active site, it should have a neutral effect on the protein activity and not disturb the native secondary structure of the protein. In instances in which the replaced amino acid is directly involved in an active site the activity of that site will be lost or altered.
  • Amino-acid scanning such as Ala-scanning
  • Ala-scanning has been successfully applied for the identification of active sites in a number of proteins, and has been performed in computer-based rational drug design methods.
  • Other amino acids particularly amino acids that have a neutral effect, such as Glycine, can also be used.
  • the amino acid scanning method is employed herein for the generation of the mutant proteins for screening for identification of sites or loci in a target protein or regions in a protein that alter a selected activity.
  • the amino acids are each replaced, one-by-one along the full-length of the protein, or one-by-one in preselected domains, such as domains that possess a desired activity or exhibit a particular function.
  • the method includes the following steps:
  • an active site is not necessarily the natural active site involved in the natural activity of a target protein, but those amino acids involved in the activities of the proteins under
  • the whole process of the 'identification of the active site(s) on the full length protein sequence requires the following sub-steps: a. Generation of a mutant library (on the gene to be evolved) in which each individual mutant contains a single mutation located at a different amino acid position and that includes a systematic replacement of the native amino acid by Ala or any other amino acid (always the same throughout the entire protein sequence); b. phenotypic characterization of the individual mutants, one-by-one and assessment of mutant protein activity;
  • HITS active site(s)
  • the active site(s) the HITS
  • those amino acids either at or surrounding the active sites such as within 1 , 2, 3, . . . 10, 20 or any selected regions, as the unitary elements of exchange and generate diversity either at or around one of those sites or as a combined diversity at several sites at a time
  • This process includes the following steps: a. Generation of a new mutant library (on the gene to be evolved) in which each individual mutant contains either single or multiple mutations located at (or surrounding) a specific active site (a HIT) position detected by the precedent aa-scan process.
  • these mutations include replacement, in each individual mutant, of the native amino acid located either at (or surrounding) the HIT position by one of all other possible amino acids, such that, in the library, and at (or surrounding) each HIT position the native amino acid has been replaced by all possible amino acids.
  • b Identification of those mutants that display an increase in protein activity, thus indicating that a new sequence at or surrounding an active site has been identified with higher activity compared to the native sequence.
  • LEADS LEADS. The process can be repeated as many times as desired, in search for new combinations of optimal amino acids at (or surrounding) the different HIT sites.
  • the process includes the steps of generating of a new mutant library (of the gene to be evolved) in which each individual mutant contains either single or multiple mutations located at (or surrounding) a specific active site (a HIT) position; phenotypic characterization of the individual mutants, one-by-one and assessment of mutant protein activity; and identification of those mutants that display an increase in protein activity, thus indicating that a new sequence at or surrounding an active site has been identified with higher activity compared to the native sequence.
  • LEADS second generation LEADS
  • Phenotypic characterization of the gene variants This step requires the expression of the gene variants in order to allow them to manifest their respective phenotypes. Gene expression can be accomplished by different means: in vitro, in reconstituted systems or in vivo in cellular systems, including bacterial and eukaryotic cells. For all exemplification purposes, reference is made to in vivo systems. Those of skill in the art can readily adapt these methods for in vitro systems, including those using biochemical assays. This step is a crucial step for several reasons:
  • variant proteins may or may not be appropriately processed, especially when post-translational modifications are involved, and therefore be able or not to elicit their potential activity. Consequently, the expression system (bacteria vs. eukaryotic cells) has to be carefully chosen.
  • the expression system (either bacteria or mammalian cells) is only the vehicle to convert the gene variants into protein variants.
  • the phenotypic characterization is performed on the protein variants, and may have nothing to do, depending on the specific system under study, with the cellular system used to express the variants.
  • the phenotypic characterization requires the use of specific assays (either biochemical (cell-free) or cell-based assays) in which the activity of the different cell mutants is challenged and assessed. In addition to the implications discussed below, these assays must be designed in such a way that they reflect the final environment in which the 'evolved' protein is expected to act.
  • the assay when optimizing an enzyme to be used in an artificial industrial setting, the assay should reproduce those conditions (temperature, pH, media composition%) of the real-life industrial reaction mixture, which may be relatively easy to do.
  • the final destination of the 'evolved' protein is a complex biological setting, such as the intracellular environment, the extra-cellular milieu (example: circulating proteins) or the structure of a virus, the necessary assay(s) may be quite difficult to setup.
  • most of the work done so far on directed evolution has been made on simple enzymes for which all the necessary settings are relatively easy to implement.
  • RTVT TM Real Time Virus Titering
  • TREE Real Time Virus Titering
  • the methods in this section are also used in data analysis when measuring the output signal. As described below, output signal can be assessed by a Hill analysis or a second order polynomial or other algorithm that describes the interaction of biological molecules in complex system. In addition, where the output signal is actually the number of viral particles or ip produced, the methods in this section RTVT and TREE are advantageously used.
  • Prior art virus titration methods for determining the amount of virus present in a biological sample, are based on the assessment of some kind of output signal, such as cytopathic effect, lysis or plaques and cell fusion focuses, induced in a reporter cell following a fixed time after infection with varying concentrations of the sample containing the virus. The lowest concentration of the sample at which no signal can be measured is taken as the titer of the virus in that sample.
  • These approaches are known as “serial dilution” or “limiting dilution” methods.
  • limiting dilution methods one single virus concentration, measured at a given time end-point gives rise to a single measurement of the output signal.
  • the cell reports an output signal in response to the infection and, therefore, it is named here a reporter cell.
  • One such output signal is, for example, the expression of the genes carried by the virus (whether they are viral genes or exogenous genes (transgenes)) .
  • the output signal (for instance the level of expression of those genes) develops over time and depends, mainly, on two factors: i) the time point ("t") at which its level is measured after infection and ii) the amount of virus infecting the cell; i.e. the concentration of the virus preparation used to infect the cell ("s").
  • the output signal at a given time point after infection, will be higher for higher concentrations of the virus infecting the reporter cells; and for any given concentration of virus, the output signal increases with time after infection until it generally reaches a plateau level.
  • Real Time Virus Titering uses non-destructive methods for the assessment of output signal.
  • Real Time Virus Titering is a viral titration method based on the kinetic analysis of the development of the output signal in virus-infected cells, tested at a single concentration of virus or biological sample. Instead of fixing the time point after infection and varying the concentration of the sample as is done in limiting dilution methods, in the Real Time Virus Titering RTVT TM method, a fixed concentration of virus is used and the generation of a signal over time is assessed.
  • the signal is measured as a function of time at a single virus concentration.
  • a single virus sample concentration
  • whose output signal is measured at a number of time points can give rise to as many measurements of the output signal as needed, and, eventually to a continuous, over time, assessment of the signal in real time.
  • Real Time Virus Titering RTVT TM can be advantageously used in high throughput methods in which large numbers of biological samples are analyzed at the same time. This is the case, for instance, when titering viruses in a virus library.
  • Limiting dilution methods rely on the output signal generated by a number of dilutions of each individual sample. If, for example, 10 dilutions (or experimental points) of each virus are used for a titration using a limiting dilution method, the analysis of a library containing 10,000 viruses require analysis of 10 5 (i.e., 10 x 10,000) experimental points.
  • the Real Time Virus Titering RTVT TM method requires only one dilution per sample, thereby requiring 10-fold fewer experimental points than a limiting dilution method.
  • the time (t ⁇ ) necessary for the output signal to reach a reference value ( ⁇ ) is a direct function of the concentration of virus.
  • t ? can be used to directly determine the concentration of the virus.
  • a limitation of the Real Time Virus Titering RTVT TM limiting dilution titering method is that not all the viruses (nor the genes carried by the viruses) generate a readily measured output signal that can be followed over time using non-destructive methods.
  • a method for titering designated Tagged Replication and Expression Enhancement Technology is provided herein.
  • This system includes: i) a cell, ii) a reporter virus carrying a reporter gene, whose activity can be followed over time by a non- destructive method (i.e., fluorescence), iii) the virus (or virus library to be titered), herein referred to as the "titering virus".
  • the elements are employed such that the virus to be titered interferes with any output signal generated by the reporter virus, leading to either decrease or increase in the amount of that signal. The higher the amount of virus to be titered, the higher is the interference with the reporter virus and output signal.
  • the kinetics of the output signal generated by the reporter virus are followed using the Real Time Virus Titering RTVT TM titering method.
  • the output signal takes longer (or shorter) to develop as a function of the amount of virus to be titered.
  • t ⁇ the time necessary for the output signal to reach a reference value ( ⁇ ) is a direct function of the concentration of virus and, therefore, t ⁇ can be used to determine the concentration of the virus to be titered.
  • the concentration (titer) of the virus to be titered can be assessed by assessing the change induced in t ⁇ by an aliquot of the virus to be titered.
  • a calibrated TREE titering assay only one aliquot virus to be titered is needed to determine its titer, which is determined by measuring the shift in the t ? of the system.
  • a calibration curve representing t ⁇ vs. the amount of virus to be titered is obtained using aliquots of a reference batch of virus of known titer (previously determined using any titering procedure) .
  • the calibration curve can then be used to determine the amount of virus in a sample of unknown titer, based on the change caused by an aliquot of the sample on the t ⁇ of the system and the corresponding titer read from the calibration curve.
  • the output signal can be assessed by a Hill analysis (see Examples and (published International PCT application No. WO 01 /44809 based on PCT n ° PCT/FR00/03503),.) or a second order polynomial (see, Examples and (Drittanti et al. (2000) Gene Ther. 7: 924- 929)) or other algorithm that describes the interaction of biological molecules in complex system, such as the interaction between cells and biological agents.
  • RTVT TM Real Time Virus Titering
  • TREETM Tagged Replication and expression enhancement
  • the process provided herein includes the following steps. 1. Generation of diversity or source of existing diversity
  • a plasmid library containing the genetic variants containing the genetic variants.
  • the genetic variants are physically separated from each other. Any model such as, but not limited to, amino acid scanning, mutagenesis, or recombination may be used to generate the plasmid library. 2. Expression of the genetic variants
  • the mutated forms of the nucleic acid are prepared or introduced into plasmids for expression in bacterial cells.
  • the genetic variants are expressed from suitable bacterial cells, which are prepared by transformation of aliquots of the cells with each member of the plasmid library (each genetic variant continues to be physically separated from each other) .
  • b. Expression in eukaryotic host cells A virus library is generated from the plasmid library.
  • the virus library, in which each different member is separately maintained, is prepared by:
  • WO 01 /1 86291 and the EXAMPLES below or a refinement of that method provided herein and designated Tagged Replication and expression enhancement (TREETM) described above and in the examples; (3) Standardization of the virus library to equal concentrations of all the individual viruses in the library (individual viruses continue to be physically separated from each other);
  • the variant proteins are expressed (from either plasmids in bacterial cells (step 2) or viruses in mammalian cells (step 4)) and their activity is assessed in one or more appropriate specific assays.
  • the assays can be both types: biochemical (cell-free) assays and/or cell-based assays.
  • the variant proteins in the library are physically separated from each other and their activity is individually assessed on a one-by-one basis.
  • the assays can be performed in one of a variety of ways, including, but are not limited to: a.
  • each individual protein variant is individually tested for the parameters that assess the activity, property, function or structure of interest. Variants are ranked out according to their activity features. Those variant proteins best suited for the specificities of each individual project and system under study are then selected. The selected leads can be used for the desired purpose or further evolved or mutated to achieve desired activities. Typically, as for most directed evolution methods, the process is an iterative one, in which mutated variants are produced, screened, the best identified and then selected. The selected variants are then subjected to further evolution and the screening process repeated. This is repeated until the desired goal is achieved. This further evolution may employ the methods herein or any directed evolution method or combinations thereof. The methods for variant production will include the amino-acid scan method herein, which provides a rational approach to variant generation. Other rounds can include combinations of any other method for directed evolution known and/or combinations thereof.
  • Recombinant viruses have been developed for use as gene therapy vectors. Gene therapy applications are hampered by the need for development of vectors with traits optimized for this application. The high throughput methods provided herein are ideally suited for development of such vectors. In addition to use for development of recombinant viral vectors for gene therapy, these methods can also be used to study and modify the viral vector backbone architecture, trans- complementing helper functions, where appropriate, regulatable and tissue specific promoters and transgene and genomic sequence analyses.
  • Recombinant AAV rAAV is a gene therapy vector that can serve as a model for application of the methods herein for these and other purposes.
  • Adeno-associated virus is a defective and non-pathogenic parvovirus that requires co-infection with either adenovirus or herpes virus, which provide helper functions, for its growth and multiplication.
  • AAV Adeno-associated virus
  • AAV are members of the family Parvoviridae and are assigned to the genus Dependovirus. Members of this genus are small, non-enveloped, icosahedral with linear and single-stranded DNA genomes, and have been isolated from many species ranging from insects to humans.
  • AAV can either remain latent after integration into host chromatin or replicate following infection. Without co-infection, AAV can enter host cells and preferentially integrate at a specific site on the q arm of chromosome 1 9 in the human genome.
  • the AAV genome contains 4975 nucleotides and the coding sequence is flanked by two inverted terminal repeats (ITRs) on either side that are the only sequences in cis required for viral assembly and replication.
  • ITRs contain palindromic sequences, which form a hairpin secondary structure, containing the viral origins of replication.
  • the ITRs are organized in three segments: the Rep binding site (RBS), the terminal resolution site (TRS), and a spacer region separating the RBS from the TRS.
  • AAV genes are complex and involves positive and negative regulation of viral transcription.
  • the regulatory proteins Rep 78 and Rep 68 interact with viral promoters to establish a feedback loop (Beaton et al. (1 989) J. Virol 53:4450-4454; Hermonat (1 994) Cancer Lett 37:129-136). Expression from the p5 and p1 9 promoters is negatively regulated in trans by these proteins.
  • Rep 78 and 68 which are required for this regulation, have bind to inverted terminal repeats (ITRs; Ashktorab et al. (1 989) J. Virol. 53:3034-3039) in a site- and strand-specific manner, in vivo and in vitro.
  • the genome (see, FIG. 4) is organized into two open reading frames (ORFs, designated left and right) that encode structural capsid proteins (Cap) and non-structural proteins (Rep). There are three promoters: p5 (from nucleotides 255 to 261 : TATTTAA), p19 (from nucleotide 843 to 849: TATTTAA) and p40 (from nucleotides 1822 to 1827: ATATAA).
  • the right-side ORF (see FIG. 4) encodes three capsid structural proteins (Vp 1 -3).
  • Capsid proteins VP1 , VP2 and VP3 initiate from the p40 promoter.
  • VP1 uses an alternate splice acceptor at nucleotide 2201 ; whereas VP2 and VP3 are derived from the same transcription unit, but VP2 use an ACG triplet as an initiation codon upstream from the start of VP3.
  • two promoters p5 and p19 direct expression of four regulatory proteins.
  • the left flanking sequence also uses a differential splicing mechanism (Mendelson et al. (1986) J. Virol 50:823-832) to encode the Rep proteins, designated Rep 78, 68, 52 and 40 on the basis molecular weight.
  • Rep 78 and 68 are translated from a transcript produced from the p5 promoter and are produced from the unspliced and spliced form, respectively, of the transcript.
  • Rep 52 and 40 are the translation products of unspliced and spliced transcripts from the p19 promoter.
  • the rep protein is a adeno-associated virus protein involved in a number of biological processes necessary to AAV replication.
  • the production of the rRep proteins enables viral DNA to replicate, encapsulate and integrate (McCarty et al. (1 992) J. Virol 55:4050-4057; Horer et al. ( 1 995) J. Virol 53:5485-5496, Berns et al. ( 1 996) Biology of Adeno-associated virus, in Adeno-associated virus (AAV) Vectors in Gene Therapy, K.I. Berns and C. Giraud, Springer ( 1 996); and Chiorini et al.
  • AAV and rAAV have many applications, including use as a gene transfer vector, for introducing heterologous nucleic acid into cells and for genetic therapy. Advances in the production of high-titer rAAV stocks to the transition to human clinical trials have been made, but improvement of rAAV production will be complemented with special attention to clinical applications of rAAV vectors as a successful gene therapy approach.
  • Productivity of rAAV i.e. the amount of vector particles that can be obtained per unitary manufacturing operation
  • Methods for high throughput production and screening of rAAV have been developed (see, e.g. , Drittanti et al.
  • the plasmid preparation, transfection, virus productivity and titer and biological activity assessment are intended to be performed in an automatable high throughput format, such as in a 96 well (or other number or multiples thereof, such as 384, 1 536 . . . 9600, 9984 . . .) formats.
  • Rep protein Since the Rep protein is involved in replication it can serve as a target for increasing viral production. Since it has a variety of functions and its role in replication is complex, it has heretofore been difficult to identify mutations that result in increase viral production.
  • the methods herein which rely on in vivo screening methods, permit optimization of its activities as assessed by increases in viral production.
  • the amino acid positions on the rep proteins that are relevant for rep proteins activities in terms of AAV or rAAV virus production are provided. Those amino acid positions are such that a change in the amino acid leads to a change in protein activity either to lower activity or increase activity.
  • the alanine or amino acid scan revealed the amino acid positions important for such activity (i.e. hits). Subsequent mutations produced by systematically replacing the amino acids at the hit positions with the remaining 1 8 amino acids produced so-called "leads" that have amino acid changes and result in higher virus production. In this particular example, the method used included the following specific steps. Amino acid scan
  • each amino acid position hit by the Ala-scan step was mutated by amino acid replacement of the native amino acid by the remaining 18 amino acids, using site-directed mutagenesis.
  • each mutant was individually designed, generated and processed separately, and optionally in parallel with the other mutants. Neither combinatorial generation of mutants nor mixtures thereof were used in any step of the method.
  • each plasmid contained a different mutant bearing a different amino acid at a different hit position.
  • each resulting mutant rep protein was then expressed and the amount of virus it could produce measured as indicated below.
  • the relative activity of each individual mutant compared to the native protein is indicated in FIGURE 3B.
  • LEADS are those mutants that lead to an increase in the activity of the protein (in the example: the ten mutants with activities higher, typically between 6 to 10 times, than the native activity).
  • the rep protein acts as an intracellular protein through complex interaction with a molecular network composed by cellular proteins, DNA, AAV proteins and adenoviral proteins (note: some adenovirus proteins have to be present for the rep protein to work).
  • the final outcome of the rep protein activity is the virus offspring composed by infectious rAAV particles. It can be expected that the activity of rep mutants would affect the titer of the rAAV virus coming out of the cells.
  • a mammalian cell-based expression system as well as a mammalian cell- based assay was used.
  • the individual rep protein variants were expressed in human 293 HEK cells, by transfection of the individual plasmids constituting the diverse plasmid library. All necessary functions were provided as follows:
  • AV adenovirus
  • AV helper functions A library of recombinant viruses with mutant rep encoding genes was generated. Each recombinant, upon introduction into a mammalian cell and expression resulted in production of rAAV infectious particles. The number of infectious particles produced by each recombinant was determined in order to assess the activity of the rep variant that had generated that amount of infectious particles.
  • the number of infectious particles produced was determined in a cell-based assay in which the activity of a reporter gene, in the exemplified embodiment, the bacterial lacZ gene, or virus replication (Real time PCR) was performed to quantitatively assess the number of viruses.
  • the limiting dilution (titer) for each virus preparation was determined by serial dilution of the viruses produced, followed by infection of appropriate cells (293 HEK or HeLa rep/cap 32 cells) with each dilution for each virus and then by measurement of the activity of the reporter gene for each dilution of each virus.
  • Hill plots NAUTSCAN TM ) as described herein (published as International PCT application No.
  • WO 01 /44809 based on PCT n° PCT/FR00/0350.3, Dec, 2000; see EXAMPLES) or a second order polynomial function (Drittanti et al. (2000) Gene Ther. 7: 924-929) was used to analyze the readout data and to calculate the virus titers. Briefly, the titer was calculated from the second order polynomial function by non-linear regression fitting of the experimental data. The point where the polynomial curve reaches its minimum is considered to be the titer of the rAAV preparation. A computer program for calculation of titers has been developed (see Drittanti et al. (2000) Gene Ther. 7: 924-929) to assess the minima.
  • TREE method described herein can be used to analyze the readout data and to calculate the virus titers. The results are shown in the
  • Rep 78 is encoded by nucleotides 321 - 2, 1 86;
  • Rep 68 is encoded by nucleotides 321 -1 906 and 2228-2252;
  • Rep 52 is encoded by nucleotides 993-21 86, and
  • Rep 40 is encoded by amino acids 993-1 906 and 2228-2252 of wildtype AAV.
  • mutations that may have multiple effects. Since the Rep coding region is quite complex, some of the mutations may have several effects. Amino acids 542, 598, 600 and 601 , which are in the Rep 68 and 40 intron region, are also in the coding region of Rep 78 and 52. Codon 630 is in the coding region of Rep 68 and 40 and non coding region of Rep 78 and 52. Mutations at 10, 86, 101 , 334 and 519 have been previously identified, and mutations, at loci 64, 74, 88, 175, 237, 250 and 429, but with different amino acid substitutions, have been previously reported. In all instances, however, the known mutations reportedly decrease the activity of Rep proteins. Among mutations described herein, are mutations that result in increases in the activity the Rep function as assessed by detecting increased AAV production. Lead identification.
  • each individual rep variant was assigned a specific activity. Those variant proteins displaying the highest titers were selected as leads and are used to produce rAAV.
  • rAAV and Rep proteins that contain a plurality of mutations based on the hits (see Table in the EXAMPLES, listing the hits and lead sites), are produced to produce rAAV and Rep proteins that have activity that is further optimized. Examples of such proteins and AAV containing such proteins are described in the EXAMPLES.
  • the rAAV rep mutants are used as expression vectors, which, for example, can be used transiently for the production of recombinant AAV stocks.
  • the recombinant plasmids may be used to generate stable packaging cell lines.
  • the recombinant vectors expressing the AAV with mutant rep genes for example, are cotransfected into host cells with a plasmid expressing the neomycin phosphotransferase gene (neor) by transfection methods well known to those skilled in the art, followed by selection for G418 resistance.
  • rAAV gene therapy for the purpose of transferring genetic information into appropriate host cells for the management and correction of human diseases including inherited and acquired disorders such as cancer and AIDS.
  • the rAAV can be administered to a patient at therapeutically effective doses.
  • a therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of disease.
  • Toxicity and therapeutic efficacy of the rAAV can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDS 50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) .
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Doses that exhibit large therapeutic indices are preferred. Doses that exhibit toxic side effects may be used; care should be taken to design a delivery system that targets rAAV to the site of treatment in order to minimize damage to untreated cells and reduce side effects.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such rAAV lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (ie., the concentration of the test compound which achieves a half-maximal infection or a half- maximal inhibition) as determined in cell culture.
  • IC 50 ie., the concentration of the test compound which achieves a half-maximal infection or a half- maximal inhibition
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • RTVTTM Real Time Vector Titering
  • This Example is based on the method described in International PCT application No. PCT/FR01 /01 366, based on French application n° 0005852, filed 9 May 2000, and published as International PCT application No. WO 01 /1 86291 .
  • This method assesses the titer or concentration of a biological agent (virus, gene transfer vector) in a sample, by measurement of the kinetics of change of a reporter parameter following the exposure of cells to the biological agent.
  • reporter parameters may include, but are not limited to,: gene / transgene expression related to the gene/transgene products, such as enzymatic activity, fluorescence, luminescence, antigen activity, binding to receptors or antibodies, and regulation of gene expression), differential gene expression, viral/vector progeny productivity, toxicity, cytotoxicity, cell proliferation and/or differentiation activity, anti-viral activity, morphogenetic activity, pathogenetic activity, therapeutic activity, tumor suppressor activity, oncogenetic activity, pharmacological activity.
  • gene / transgene expression related to the gene/transgene products such as enzymatic activity, fluorescence, luminescence, antigen activity, binding to receptors or antibodies, and regulation of gene expression
  • differential gene expression viral/vector progeny productivity, toxicity, cytotoxicity, cell proliferation and/or differentiation activity, anti-viral activity, morphogenetic activity, pathogenetic activity, therapeutic activity, tumor suppressor activity, oncogenetic activity, pharmacological activity.
  • Serial dilution methods The assessment of the concentration or titer of biological agents using current approaches needs serial dilutions of the agent. Serial dilutions of the agent are applied to a cell-based reporter system, that elicits an output signal in response, to the exposure to the agent. The intensity of the signal is a function of the concentration of the agent. The titer or concentration of the agent is determined as the highest dilution that still elicits a measurable response in the output. The higher the number of dilutions tested, the higher the accuracy of the value obtained for the titer. This approach requires a set of serial dilutions for every biological agent whose titer needs to be determined. Thus, the application of this approach to the simultaneous titration of a large number of different biological agents is limited by the number of experimental points needed
  • the intensity of the output signal (after exposure of reporter cells to the biological agent) is not only dependent on the concentration of the agent but also on the time after exposure. As time increases, the intensity of the signal increases. The kinetics of change of the intensity over time depends upon the concentration of the agent. Thus, lower concentrations of the agent will require longer times for the intensity to reach a given value that would be reached in shorter times after exposure to higher concentrations of the same agent.
  • RTVT TM Real Time Virus Titering
  • the system includes the following elements: a preparation of the biological agent (virus, gene transfer vector, protein,...) whose concentration or titer is unknown and has to be determined. a reporter system including culture of a cell line (or a mixture of cell lines) that reacts to the exposure to the biological agent by displaying a specific output signal, a master preparation of a reference biological agent, of known concentration or titer, that is able to generate the output signal when the reporter cells are exposed to it.
  • the intensity of the output signal is called /; the concentration of the biological agent used is called c, the time of exposure of the cells to the biological agent is called t.
  • the intensity of the output signal (/ ) is a function of c and f : / increases as the concentration (c ) of the biological agent applied to the cells increases;
  • ⁇ is defined here as a threshold value of the intensity of the output signal, arbitrarily defined for every system under study.
  • t ⁇ is defined here as the time necessary to reach the threshold ⁇ .
  • the reporter cells are exposed to serial dilutions of a reference biological agent, whose concentration (or titer) is previously known.
  • the intensity of the output signal (/) is measured at several time points (t) for every concentration (serial dilutions) c of the reference biological agent.
  • This plot represents the time necessary for the intensity of the output signal to. reach the threshold value ⁇ as a function of the concentration of the biological agent used. This is a standard plot and will be used to determine the unknown concentration of the biological agent under study by measuring the time that a given dilution of it needs to give an output signal whose intensity equals the threshold ⁇ .
  • the reporter cells are exposed to a dilution of the biological agent under study (whose concentration or titer is to be determined) .
  • the intensity of the output signal ( ) is measured over time until it reaches the threshold value ⁇ .
  • the time necessary for / to reach the value ⁇ is recorded as t ⁇ .
  • This c value represents the concentration or titer of the biological agent under study.
  • Rat-2 cells were infected with serial dilutions of a reference preparation of a retroviral vector carrying the green fluorescent protein (GFP) gene (vector pSI-EGFPI see, Ropp et al. (1 995) Cytometry 27 :309- 31 7). At increasing times after infection, the level of expression of the transgene was determined (as the level of fluorescence due to the GFP gene) as the output signal.
  • Table 3 represents the values obtained:
  • the threshold value of ⁇ 100 was arbitrarily selected for this example.
  • the time (t ⁇ ) necessary for the output signal to reach the threshold ⁇ , for every concentration is shown in table 4.
  • a plot of t ⁇ versus concentration for the reference virus shows that the concentration and t ⁇ exhibit a clearly defined relationship, that allows for the calculation of the concentration (c) of a sample, if the corresponding t ⁇ of that sample is known.
  • TREE is a method for titering and standardization of preparations of viruses, vectors, antibodies, libraries, proteins, genes and any other moiety that is detectable based upon a output signal, such as fluorescence.
  • the TREE method is an improvement of the Real Time Virus Titering (RTVT) method (see, International PCT application No. PCT/FR01 /01366 published as International PCT application No. WO 01 /186291 ). It is performed with a reporter moiety, such as a reporter virus (with a known titer) and the test sample (with unknown titer).
  • RTVT Real Time Virus Titering
  • the reporter such as a reporter virus
  • the reporter has a readily detectable output signal that can be measured as a function of time.
  • the effect of the moiety, such as a virus, of unknown titer is assessed.
  • the moiety whose titer is assessed either increases or decreases the output signal as a function of time. This change in signal is used to assess the amount or concentration of the moiety of unknown concentration, and hence its titer.
  • the method is exemplified herein using an AAV system for the determination of the titer of an AAV vector and an AAV-reporter vector as a competitor and wild type Adenovirus as helper virus.
  • AAV AAV-reporter vector
  • One of skill in the art readily can adapt the method to other systems, including other viruses, and other moieties for which a reporter system can be developed.
  • Other such moieties include, but are not limited to, viral vectors, plasmids, libraries, proteins, antibodies, vaccines, genes, and nucleic acid molecules.
  • HeLa rep-cap32 cells a HeLa derived cell line (kindly provided by P. Moullier, Laboratoire de Therapie Genique, CHU, France; see, Salvetti et al. (1 998) Hum Gene Ther 20:695-706; Chadeuf et al. (2000) J Gene Med 2:260-268) was grown in DMEM with 10% fetal calf serum. These cells were plated 24 h before infection at a density of 1 x 10 4 cells in single well of 96-well plates.
  • Hela rep-cap32 cells had been produced by cotransfecting plasmid pspRC, which harbors the AAV rep-cap genome with the ITRs deleted (bp 1 90 to 4484 of wild-type AAV), with plasmid PGK-Neo, conferring resistance to G418 on Hela cells (see, Chadeuf et al. (2000) J. Gene Med. 2:260-268 and Salvetti et al. ( 1 9981 Hum Gene Ther. 3:695-706). Hela rep-cap 32 cells are a packaging line that harbor one copy of the genome with the ITRs deleted (see, also Tessier et al. (2001 ) J. Virol. 75/375-383).
  • Plasmid pspRC contains the AAV genome (positions 190-4,484 bp) with the ITRs deleted and was obtained by excising the rep-cap fragment (Xha ⁇ fragment) from the well-known vector psub201 (Samulski et al. (1987) J Virol 57:3096-3101 ; also called pSSV9) by Xba ⁇ digestion and inserting it into the Xba ⁇ site of plasmid pSP72 (Promega).
  • Plasmid psub201 (see, e.g., U.S. Patent No. 5,753,500) is a modified full-length AAV type 2 genomic clone contains all of the AAV type 2 wild-type coding regions and cis acting terminal repeats. 2.
  • rAAV-GFP is an SSV9-derived vector; SSV9 is a clone containing the entire adeno-associated virus (AAV) genome inserted into the Pvull site of plasmid pEMBL (see, Du et al. (1996) Gene Ther 3:254-261 ).
  • the rAAV-GFP and rAAV-LacZ plasmids are SSV9 with a GFP or LacZ gene under control of the cytomegalovirus (CMV) immediate-early promoter. All the samples were done in triplicate. 3. Process
  • Figure 2 shows the overall procedure in 96 well format.
  • Cells were plated 24 h before infection.
  • Ad5 different MOD, were done.
  • the plates were read at different times using the Analyst AD&HT micro plate-reader (LJL BioSystems).
  • Fluorescence Intensity (FI) of the infected cells is measured as a function of the time.
  • Serial dilutions of the AAV- competitor vector AAV-lacZ vector, which decreases the fluorescence signal, are performed.
  • fluorescence was measured for AAV-GFP with 10 6 ip and 10 5 ip and then 10 6 ip of the AAV-GFP reporter virus in the serial dilutions of the competitor virus, AAV-lacZ vector in a 96-well format (samples 1 -4, see Table below). Measurements were taken of each well and curves of FI (of the GFP) versus time (hrs) were obtained (see Figure 2B).
  • the difference between 10 6 ip of rAAV-GFP put in each sample and the number of ip detected by fluorescence in the same well is the actual quantity of rAAV-GFP competed (consumed) by the unknown rAAV (in this case rAAV-LacZ) . This number is determined for each dilution.
  • the quantity rAAV-GFP consumed is the same quantity of unknown rAAV in the sample. This quantity is present in one volume of unknown rAAV, which in this example is 1 ml. Based upon this, the infectious titer of the unknown rAAV is determined. The results are shown in Table 2.
  • e average titer using t s met od was 6.
  • x 10 7 ip/ ⁇ l 6.01 x 10 5 ip/0.01 ⁇ l).
  • the standard deviation was 1 .37 x 10 10 ip/ml with an error of ⁇ 23%.
  • Two widely used parameters that provide quantitative information about the potential performance of a gene transfer vector preparation are the titer of physical particles and the titer of infectious particles.
  • Vector preparations with high titer of infectious particles and low physical particles/infectious particles ratio are considered to be of higher quality.
  • the titer in physical particles(p ⁇ ) see, e.g., Mittereder et al. (1996) J. Virol. 70:7498-7509; Atkinson et al. (1998) Nucl. Acids Res. 25:2821 - 2823; Kechli et al. (1998) Hum. Gene Ther. 3:587-590; and Nelson et al. (1998) Hum. Gene Ther.
  • nucleic acid contents nucleic acids hybridization and OD 260 respectively for AAV and AdV
  • viral protein content for example, reverse transcriptase (RT) activity and p24 content for MLV and HIV, respectively.
  • pp there are particles potentially active in performing transduction (ip, infectious particles), as well as particles that are inactive (nip, non-infectious particles) (Ruffing et al. (1994) J. Gen. Virol. 75:3385-3392; Kechli et al. (1998) Hum. Gene Ther. 3:587-590.).
  • the pp and the ip/nip ratio are features of the packaging system, the manufacturing process and the vector itself.
  • the infectious particles (ip) infectious units, transducing units, etc.
  • Infectious particles (ip) measures the number of particles effective in performing a process whose output is being measured; not all particles participate or are capable of participating in all processes.
  • ip measures the number of active particles in the measured process; it includes physical particle (pp) and inactive particles (nip or non-infectious particles) .
  • pp physical particle
  • inactive particles nip or non-infectious particles
  • biological agents viruses, vectors or cells
  • a number of useful parameters, derived from the Hill equation, are scored and used to quantify relevant features of the biological agent, of the cells, as well as of the biological process or reaction involved.
  • the Hill-based analysis ( ⁇ t, ⁇ , ⁇ ,6, ⁇ , ⁇ ) is used for a variety of purposes, including, but not limited to: i) validation and optimization of the manufacturing processes used to obtain the biological agents; ii) development and optimization of the components of the biological agents (proteins, genomes, genetic units); iii) development and optimization of assays and analytical tests for the characterization of the biological agents.
  • the method includes the steps of:
  • R1 represents the biological agent concentration in a sample from the scale
  • R2 is concentration of target cells (in vitro or in vivo)
  • P (output) represents the product from the reaction R1 + R2 at a t moment;
  • P max represents the reaction maximal capacity;
  • K represents, at a constant R2 concentration, the resistance of the biological system for responding to the biological agent (resistance constant R2); r represents a coefficient that depends on R1 and corresponds to the Hill coefficient; and ⁇ represents the intrinsic power of the R1 biological agent to induce a response in the biological system (P production at the t moment), and
  • the activity of a biological agent on a complex biological system as well as its intrinsic features can be fully characterized and compared.
  • different biological systems either in vitro cell-based) or in vivo can be compared.
  • R1 r / (K + (R1 ) r ) R2 constant (1 ) r 1
  • R1 , P, P max and K represent, respectively, the concentration of the reagent R1 , the concentration of the product, the maximal capacity of the reaction and the 'affinity' constant between R1 and R2.
  • the Hill's coefficient (r) is a function of R1 .
  • the coefficient r is equal to 1 when independent non-interactive binding sites are involved between R1 and R2, such as in reactions that follow kinetics described by Michaelis- Menten; and r varies from 1 to n for systems where the sites involved in the interaction between the R1 and R2 are not independent from each other, and the affinity for R1 at any R2 binding site varies as a function of either i) the degree of occupancy of other R2 sites; ii) the concentration of R1 itself or iii) the concentration of other (positive or negative) regulators.
  • the Hill equation thus, is a general formalization that describes the interaction reaction between molecules. It expresses the amount of product formed as a function of the concentration of the reagents and of the affinity constant of the system. Originally developed for the study of the dissociation between haemoglobin and oxygen, the Hill equation covers the formal Michaelis-Menten analysis of enzyme kinetics, the analysis of ligand-receptor binding and of the allosteric protein systems.
  • the Hill equation describes the accumulation of the product P as a function of the concentration of one of the reagents (R1 ) and of the intrinsic properties (K) of the system.
  • This equation can be applied to complex biological systems. For example, the response of the cells to infection (P), can be analyzed by applying an Hill-type equation. The amount of cells growing in vitro (R2) are infected with increasing concentrations of recombinant viruses (R1 ), and (P) is monitored. A Hill equation is iteratively fitted to the experimental data.
  • P, P max , R1 , ⁇ , r and K represent, respectively, the output signal (P) (the level of viral gene expression, or the level of virus replication), the maximal output signal (Pmax), the initial concentration (R1 ) of infectious viral particles (those susceptible to trigger the process leading to P), the potency of the vector ( ⁇ ; a factor that affects the concentration of the vector (R1 ) by its specific strength or activity, the Hill's coefficient (r) and the constant of resistance of the reaction or process (K) .
  • K The constant of resistance K
  • K is analogous to those of dissociation, kinetics, equilibrium or affinity constants concepts for simple chemical and biological reactions
  • K is a feature of the process (reaction) and of the biological system tested (cell type)
  • K is a key parameter for the characterization of the assay system and the assessment of its performance as a test for the reaction under study.
  • K measures the internal resistance offered by the process or reaction triggered by the biological agent, to proceed to P.
  • K is specific to a particular process or reaction tested.
  • K is specific to the particular biological system tested. Different cell lines and types will display different K for the same reaction. Moreover, factors affecting the performance of a cell to accomplish the reaction (like contaminants, toxic agents, etc.) affect K in that cell.
  • Variations in K affect equation (2) by shifting the curve to the right or to the left, according to whether the value of K increases or decreases, respectively. All curves differing only in K are parallel each other.
  • K finds its direct and practical application in i) assay development and validation and ii) assessment of the susceptibility or sensitivity of different cell types or tissues to undertake the reaction under study and to be affected by it.
  • the potency ⁇ ⁇ measures the intrinsic potency of the biological agent to accomplish P against the resistance (K) offered by the reaction process. For every infectious virus particle (R1 ) added to the assay, the actual activity of the virus added is given by ⁇ rR1 . In order to report an output P, the potency ⁇ has to push forward the reaction inside the cell against K.
  • ⁇ is specific to the particular biological agent for the reaction under study.
  • ⁇ is a feature of the biological agent.
  • ⁇ is analogous to that of chemical activity by opposite to concentration for simple compounds, ⁇ is a correction factor that affects the concentration (R1 ) of the biological agent to indicate its actual strength or activity for a given reaction process.
  • ⁇ is a key parameter for the characterization of the biological agent and the assessment of its performance to accomplish the reaction under study. 77 finds its direct and practical application in i) biological agent optimization and development as it allows to compare the relative potency of variants of the agent. n is a valuable tool in the field of vaccine, gene transfer vector and antibody development, for the comparison between two or more different agents or different versions of the same agent, for performance.
  • n a quantitative and accurate parameter for assessing potency, will allow for ranking the candidates according to their potency (i.e., for gene transfer, gene expression, immunogenicity and other such properties and activities) and to make rational decisions about the relative value of the agent leads.
  • the efficiency e e measures the maximal global efficiency of the reaction process when a biological agent characterized by a given ⁇ value interacts with a biological system characterized by a given K.
  • e is specific to the particular couple biological agent ( ⁇ ) I biological system (K) for the reaction under study, e is a feature of the global reaction process and intervening reagents. Changes in either ⁇ , K, or both, will lead to changes in e.
  • the efficiency of the reaction process described by equation (2) is given by the increase in the output P that can be obtained by increasing the input R1 .
  • the first derivative of P with respect to R1 or the slope of the curve described by equation (2), gives the global efficiency of the reaction at every R1 input.
  • the maximal global efficiency, or e is given directly by either the slope at the inflection point of the curve described by equation (2) or by the maximum of its derivative c5P/ ⁇ 5R1 .
  • the slope of the curve given by equation (2) and the maximum of c5P/ R1 increase as e increases.
  • e is a key parameter for the characterization of the efficiency global process, considering the assay conditions and reagents all together. It is therefore useful for assay optimization once ⁇ and K have been fixed and to detect changes in n when K is kept constant or, inversely, changes in K while ⁇ is kept unchanged.
  • the heterogeneity index ⁇ ⁇ measures the internal heterogeneity of the reaction process under study.
  • Complex processes include a huge chain of individual and causal events inside a multidimensional network of interrelated and interregulated biological reactions.
  • the presence of internal heterogeneity in the reaction process can be detected by the appearance of steps in the rate of change of the Hill coefficient, corresponding to the Hill curve that fits the experimental data.
  • ⁇ is a key parameter for the dissection and detailed analysis of the reaction process. It is useful for the independent optimization and development of every one of the steps identified by ⁇ .
  • the presence of steps in the rate of change of ⁇ translates in an abrupt discontinuity in K. Therefore, every step is determined by a different macroscopic constant of resistance K.
  • One part of the curve is described by ⁇ l and the other by ⁇ 2.
  • the fact that P does not exist for R1 below R1 m ⁇ n means that there is no 'product' when the concentration of 'substrate' is lower than R1 min ; e.g. that the system is not responsive to concentrations below R1 m ⁇ n .
  • the minimal concentration of R1 that the system can detect and report is R1 m ⁇ n .
  • R1 m ⁇ n represents the minimal amount that can elicit a response in a given reporter system, and it is represented by T.
  • the titer defined this way, is neither an asymptote value nor a value approached by extrapolation, but a precise parameter of the Hill equation, at the very mathematical origin of the curve.
  • r measures the limiting dilution or apparent titer of the biological reagent. The value of r is determined by the limit of sensitivity of the biological assay system and of method used for the measurement of the product P; that is why it is said to be apparent titer.
  • T is specific to the batch or stock of the biological reagent tested
  • r represents the apparent concentration of the biological agent and is expressed in units per volume, e.g. the maximal dilution of the biological agent that leads to the production of P.
  • r is given by the maximal R1 for which the Hill coefficient reaches its minimal value (the Hill coefficient becomes constant at a value equal or close to 1 ).
  • the concept of r corresponds to that of titer, of general use for viruses, antibodies and vectors.
  • Variations in ⁇ affect equation (2) by shifting the curve to the right or to the left, according to whether the value of T decreases or increases, respectively.
  • r is a key parameter that measures the 'apparent' concentration of a stock of the biological agent, which is necessary for whatever use it will be given.
  • the absolute titer ⁇ ⁇ is a the parameter that measures the absolute concentration (titer) of a stock or batch of the biological agent.
  • the value of ⁇ is not determined by nor dependent on the limit of sensitivity of the biological assay system or of the method used for the measurement of the product P; that is why it is said to be absolute titer.
  • ⁇ is specific to the batch or stock of the biological reagent tested. It represents the real physical concentration of the biological agent and is expressed in units per volume, e.g. the maximal dilution of the biological agent that leads to the production of P.
  • the ratio of the absolute titer ⁇ corresponding to two biological agent preparations, can be obtained from their respective ⁇ and T.
  • Variations in ⁇ affect the equation (2) by shifting the curve to the right or to the left and/or by changing its slope.
  • Compensation between and K ⁇ and K may appear to compensate to generate two different Hill curves (one differing in 77 and the other one differing in K) that would apparently fit with the same experimental data.
  • two Hill curves in which the increase in 77 is compensated by the decrease in K, and vice versa, may seem to represent the same curve, which could make it difficult to determine whether two Hill curves are different because a change in ⁇ or in K.
  • the structure of the potential drug in this case the biological agent
  • the biological agent must be optimized to a maximal possible intrinsic potency.
  • the goal is to search for better performing reporter systems (the lowest possible K) , as analytical tool.
  • Two different systems characterized by constants ⁇ and /cB, respectively, can be compared (using the same biological agent) for their relative resistance or performance.
  • Complex systems involving the interaction of biological agents, such as viruses, vaccines, gene transfer vectors, antibodies proteins and living cells can be analyzed using the Hill equation.
  • a complex succession of unitary processes, each of them susceptible to be individually analyzed by the Hill equation, as a global process, can be also described by the same equation as its constitutive steps.
  • HEK human embryo kidney cells
  • ATCC Dulbecco's modified Eagle's medium containing 4.5 g/l glucose (DMEM; GIBGO-BRL) 10 % fetal bovine serum (FBS, Hyclone).
  • DMEM Dulbecco's modified Eagle's medium containing 4.5 g/l glucose
  • FBS % fetal bovine serum
  • Hela rep-cap 32 cells described above, were obtained from Anna Salvetti (CHU, France) and cultured in the medium described above.
  • Plasmids pNB-Adeno, which encodes the entire E2A and E4 regions and VA RNA I and II genes of Adenovirus type 5, was constructed by ligating into the polylinker of multiple cloning site of pBSII KS ( + /-) (Stratagene, San Diego, USA) the Sall-Hindlll fragment (9842-1 1555 nt) of Adenovirus type 5 and the BamHI-Clal fragment (21563- 35950) of pBR325. All fragments of adenovirus gene were obtained from the plasmid pBHG-10 (Microbix, Ontario, Canada).
  • pNB-AAV encodes the genes rep and cap of AAV-2 and was constructed by ligation of Xbal-Xbal PCR fragment containing the genome of AAV-2 from nucleotide 200 to 4480 into Xbal site of polylinker MCS of pBSIIKS( + /-). The PCR fragment was obtained from pAV1 (ATCC, USA). Plasmid pNB-AAV was derived from plasmid pVA1 l, which contains the AAV genomic region, rep and cap. pNB-AAV does not contain the AAV ITR's present in pAV1. pAAV-CMV(nls)LacZ was provided by Dr Anna Salvetti (CHU, France).
  • Plasmid pAAV- CMV(nls)LacZ is derived from plasmid psub201 by deleting the rep-cap region with SnaB I and replacing it with an expression cassette harboring the cytomegalovirus (CMV) immediate early promoter (407 bp), the nuclear localized ⁇ -galactosidase gene and the bovine growth hormone polyA signal (324 bp) (see, Chadeuf et al. (2000) J. Gene Med. 2:260- 268.
  • pAAV-CMV(nls)LacZ was provided by Dr Anna Salvetti.
  • Wild type adenovirus (AV) type 5 stock originally provided by Dr Philippe Moullier (CHU, France), was produced accordingly to standard procedures.
  • 25 pmol of each mutagenic primer was placed into a 96 PCR well plate.
  • 15 ⁇ l of reaction mix (0.25 pmol of pNB-AAV), 25 pmol of the selection primer (changing one non-essential unique restriction site to a new restriction site), 2 ⁇ l of 10 X mutagenesis buffer (100 mM Tris- acetate pH7.5, 100 mM MgOAc and 500 mM KOAc pH7.5) was added into each well.
  • the samples were incubated at 98°C for 5 minutes and then immediately incubated for 5 minutes on ice. Finally, the plate was placed at room temperature for 30 minutes.
  • the primer extension and ligation reactions of the new strands were completed by adding to each sample: 7 ⁇ l of nucleotide mix (2.86 mM each nucleotide and 1 .43 X mutagenesis buffer) and 3 ⁇ l of a fresh 1 :10 enzyme dilution mix (0.025 U/ ⁇ l of native T7 DNA polymerase and 1 U/ ⁇ l of T4 DNA ligase were diluted in 20 mM Tris HCl pH7.5, 10 mM KCI, 10 mM ⁇ - mercaptoethanol, 1 mM DTT, 0.1 mM EDTA and 50% glycerol) . Samples were incubated at 37°C for 1 hour. The T4 DNA ligase was inactivated by incubating the reactions at 72°C for 15 minutes to prevent re-ligation of the digested strands during the digestion of the parental plasmid (pNB-AAV).
  • Each mutagenesis reaction was digested with restriction enzyme to eliminate parental plasmids: 30 ⁇ l solution containing 3 ⁇ l of 10X enzyme digestion buffer and 10 units of restriction enzyme were added to each mutagenesis reaction and incubated at 37 °C for at least 3 hours. 90 ⁇ l of the E. coli XLmutS competent cells (Stratagene, San Diego
  • CA cortisol
  • ⁇ -mercaptoethanol a fraction of the reactions that had been digested with restriction enzyme (1 /10 of the total volume) was added to the deep well plates. The plates were swirled gently prior to incubation on ice for 30 minutes.
  • a heat pulse was performed in a 42°C water bath for 45 seconds, the transformation mixture was incubated on ice for 2 minutes and 0.45 ml of preheated SOC medium (2% (w/v) tryptone, 0.5% (w/v) yeast extract, 8.5 mM NaCl, 2.5 mM KCI, 10 mM MgCI 2 and 20 mM glucose at pH 7) was added. The plates were incubated at 37°C for 1 hour with shaking.
  • preheated SOC medium 2% (w/v) tryptone, 0.5% (w/v) yeast extract, 8.5 mM NaCl, 2.5 mM KCI, 10 mM MgCI 2 and 20 mM glucose at pH 7
  • YT medium is 0.5% yeast extract, 0.5% NaCl, 0.8% bacto-tryptone
  • ampicillin 100 ⁇ g/ml of ampicillin
  • YT medium 0.5% yeast extract, 0.5% NaCl, 0.8% bacto-tryptone
  • Plasmid DNA isolation was performed from each mutant culture using standard procedure described in Nucleospin Multi-96 Plus Plasmid Kit (Macherey-Nagel). Five hundred ⁇ g of the resulting isolated DNA was digested with 10 units of the selection restriction enzyme in a total volume of 30 ⁇ l containing 3 ⁇ l of 10X enzyme digestion buffer for overnight at 37°C.
  • a fraction of the digested reactions (1 /10 of the total volume) were transformed into 40 ⁇ l of Epicurian coli XL1 -Blue competent cells supplemented with 0.68 ⁇ l of /3-mercaptoethanol to a final concentration of 25 mM. After heat pulse, 0.45 ml of SOC was added and the transformation mixtures were incubated for 1 hour at 37°C with shaking before to be plate on LB-ampicillin agar plates. The agar plates were incubated overnight at 37°C and the colonies obtained were picked up and grown overnight at 37°C into deep-well plates.
  • rAAV Production rAAV from each of the above wells were produced by triple transfection on 293 HEK cells. 3 x 10 4 cells were seeded into each well of 96 micro-well plate and cultured for 24 hours before transfection. Transfection was made on cells at about 70% confluency. 25 kDa PEI (poly-ethylene-imine, Sigma-Aldrich) was used for the triple transfection step.
  • AV helper plasmid pNB- Adeno
  • AAV helper plasmid pNB-AAV or a mutant clone rep plasmid
  • vector plasmid pAAV-CMV(nls)LacZ
  • Titers of rAAV vector particles were determined on HeLa rep/cap 32 cells using standard dRA (serial dilution replication assay) test. Cells were plated 24 hours before infection at a density of 1 x 10 4 cells in 96- well plates. Serial dilutions of the rAAV preparation were made between 1 and 1 x 10 s ⁇ l and used for co-infection of the HeLa rep/cap 32 cells together with wt-AV type 5 (MOI 25) . 48 hours after infection the ip were measured by real time PCR or by the quantification of biological activity of the transgene.
  • dRA serial dilution replication assay
  • Infected HeLa rep/cap 32 cells were lysed with 50 ⁇ l of solution (50 mM Hepes, pH 7.4; 1 50 mM NaCl) . After one cycle of freeze-thawing 50 ⁇ l of Proteinase K (10 mg/ml) and the lysate were incubated one hour at 55 °C. The enzyme was inactivated by incubation 10 min at 96°C.
  • Results were analyzed using the Hill equation-based analysis (designated NautScanTM; see, Patent n° 991 5884, 1 999, France; published as International PCT application No. WO 01 /44809 (PCT n° PCT/FROO/03503, Dec, 2000, see EXAMPLE above) . Briefly, data were processed using a Hill equation-based model that allows extraction of key feature indicators of performance for each individual mutant. Mutants were ranked based on the values of their individual performance and those at the top of the ranking list were selected as Leads. Results Generation of diversity.
  • each amino acid in the rep protein sequence was replaced with Alanine.
  • sets of rAAV that encode mutant rep proteins in which each differs from wild type by replacement of one amino acid with Ala were generated.
  • Each set of rAAV was individually introduced into cells in a well of a microtiter plate, under conditions for expression of the rep protein. The amount of virus that could be produced from each variant was measured as described below.
  • activity of Rep was assessed by determining the amount of AAV or rAAV produced using infection assays on HeLa Rep-cap 32 cells and by measurement of AAV DNA replication using Real Time PCR, or by assessing transgene ( ⁇ -galactosidase) expression.
  • the relative activity of each individual mutant compared to the native protein was assessed and "hits" identified. Hit positions are the positions in the mutant proteins that resulted in an alteration (selected to be at least about 20%), in this instance all resulted in a decrease, in the amount of virus produced compared to the activity of the native (wildtype) gene (see Fig. 3A).
  • each individual rep variant was assigned a specific activity. Those variant proteins displaying the highest titers were selected as leads (see Table above). Leads include: amino acid replacement of T by N at Hit position 350; T by I at Hit position 462; P by R at Hit position 497; P by L at Hit position 497; P by Y at Hit position 497; T by N at Hit position 517; G by S at Hit position 598; G by D at Hit position 598; V by P at Hit position 600. Also provided are combinations of the above mutant Rep 78, 68,
  • AAV any serotype
  • AAV-2 any serotype containing the mutation at the indicated position or corresponding position for serotypes other than AAV-2, including any set forth in the following table and corresponding SEQ ID Nos.
  • Each amino acid sequence is set forth in a separate sequence ID listing; for each mutation or combination thereof there is a single SEQ ID setting forth the unspliced nucleic acid sequence for Rep78/68, which for all mutations from amino acid 228 on, includes the corresponding Rep 52 and Rep 40 encoding sequence as well.
  • nucleic acid molecules are provided in plasmids, which are introduced into cells to produce the encoded proteins.
  • the analysis revealed the amino acid positions that affect Rep proteins activities. Changes of amino acids at any of the hit positions result in altered protein activity.
  • Hit positions are numbered and referenced starting from amino acid 1 (nucleotide 321 in AAV-2 genome), also codon 1 of the protein Rep78 coding sequence under control of p5 promoter of AAV-2: 4, 20, 22, 29, 32, 38, 39, 54, 59, 124, 125, 127, 132, 140, 161 , 163, 193, 196, 197, 221 , 228, 231 , 234, 258, 260, 263, 264, 334, 335, 337, 342, 347, 350, 354, 363, 364, 367, 370, 376, 381 , 389, 407, 41 1 , 414, 420, 421 , 422, 424, 428, 438, 440, 451 , 460,
  • the encoded Rep78, Rep68, Rep 52 and Rep 40 proteins and rAAV encoding the mutant proteins are provided.
  • the corresponding nucleic acid molecules, Rep proteins, rAAV and cells containing the nucleic acid molecules or rAAV in which the native proteins are from other AAV serotypes, including, but are not limited to, AAV-1 , AAV-3, AAV-3B, AAV-4, AAV-5 and AAV-6.
  • hit positions identified include: 10, 64, 74, 86, 88, 101 , 175, 237, 250, 334, 429 and 519.
  • nucleic acid molecules, the rAAV, and the encoded proteins in which the native amino acid at each hit position is replaced with another amino acid, or is deleted, or contains additional amino acids at or adjacent to or near the hit positions are also provided.
  • nucleic acid molecules, recombinant Rep proteins from the other serotypes and the resulting rAAV are also provided (see Figs. 5 and the above Table for the corresponding position in AAV-1 , AAV-3, AAV-3B, AAV-4, AAV-5 and AAV-6).
  • AAV adeno-associate virus
  • Rep proteins and viruses encoding such proteins that include mutations at one or more of residues 64, 74, 88, 175, 237, 250 and 429, where residue 1 corresponds to residue 1 of the Rep78 protein encoded by nucleotides 321 -323 of the AAV-2 genome, and where the amino acids are replaced as follows: L by A at position 64; P by A at position 74; Y by A at position 88; Y by A at position 175; T by A at position 237; T by A at position 250; D by A at position 429 are provided. Nucleic acid molecules encoding these viruses and the mutant proteins are also provided.
  • nucleic acid molecules produced from any of the above-noted nucleic acid molecules by any directed evolution method, including, but are not limited to, re-synthesis, mutagenesis, recombination and gene shuffling and any way by combining any combination of the molecules, i.e., one, two by one, two by two, n by n, where n is the number of molecules to be combined ( i.e. , combining all together).
  • the resulting recombinant AAV and encoded proteins are also provided.
  • nucleic acid molecules of SEQ ID Nos. 563-725 and the encoded proteins are also provided.
  • Recombinant AAV and cells containing the encoding nucleic acids are provided, as are the AAV produced upon replication of the AAV in the cells.
  • Methods of in vivo or in vitro production of AAV or rAAV using any of the above nucleic acid molecules or cells for intracellular expression of rep proteins or the rep gene mutants are provided.
  • In vitro production is effected using cell free systems, expression or replication and/ or virus assembly.
  • In vivo production is effected in mammalian cells that also contain any requisite cis acting elements required for packaging.
  • nucleic acid molecules and rAAV any serotype in which position 630 (or the corresponding position in another serotype; see Figs. 5 and the table above) has been changed. Changes at this position and the region around it lead to changes in the activity or in the quantities of the Rep or Cap proteins and/or the amount of AAV or rAAV produced in cells transduced with AAV encoding such mutants.
  • Such mutations include tgc to gcg change (SEQ ID No. 721 ). Mutations at any position surrounding the codon position 630 that increase or decrease the Rep or Cap proteins quantities or activities are also provided.
  • rAAV any serotype
  • rAAV any serotype
  • In vitro methods include cell free systems, expression or replication and/or virus assembly.
  • rAAV and other serotypes with corresponding changes
  • nucleic acid molecules encoding an amino acid replacement by N at Hit position 350 of AAV- 1 , AAV-3, AAV-3B, AAV-4 and AAV-6 or at Hit position 346 of AAV-5; by I at Hit position 462 of AAV-1 , AAV-3, AAV-3B, AAV-4 and AAV-6 or at Hit position 458 of AAV-5; by either R, L or Y at Hit position 497 of AAV-1 , AAV-3, AAV-3B, AAV-4 and AAV-6 or at Hit position 493 of AAV-5; by N at Hit position 517 of AAV-1 , AAV-3, AAV-3B, AAV-4 and AAV-6 or at Hit position 535 of AAV-5; by S at hit position 543 of AAV-1 and AAV-6 or at hit position 542 of AAV-3, AAV-3B and AAV-4 or at hit position 561 of AAV-5; by S at hit position 549 of AAV-1 and
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