JP2001504325A - A method for optimization of gene therapy by recursive sequence shuffling and selection - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for evolving nucleic acids for use in gene therapy by recursive sequence combination. Many of the methods evolve vectors (both viral and non-viral vectors) to have improved properties. For example, a vector may have a viral titer, infectivity, expression of a gene in the vector, tissue specificity, viral genome capacity, episomal retention, loss of immunogenicity of the vector or its expression product, site-specific integration, increased Evolve to have improved properties such as stability or the ability to confer cell resistance to microbial infection. The present invention further provides phagemid adenoviruses capable of producing more than 10 kilobases of single-stranded DNA, including adenovirus and phage f1 origin of replication.

Description

DETAILED DESCRIPTION OF THE INVENTION   A method for optimization of gene therapy by recursive sequence shuffling and selection   This application was filed on September 27, 1996, and issued under 35 U.S.C. §111 (b) U.S.A. changed to Provisional Application No. 60 / 037,742 under 37th §1.53 (b) (2) US Patent Application No. (“USSN”) 08 / 721,824; and USSN filed September 27, 1996 A continuation-in-part of 08 / 722,660. Each of the above-mentioned applications is hereby incorporated by reference. For their entirety and all purposes, they are expressly incorporated by reference.                                Field of the invention   The present invention relates to the modification of vectors and other nucleic acids for use in gene therapy. Applied in the field of molecular genetics for good. Improvements achieved by recursive sequence recombination Is done.                       Background and description of related technologies   Gene therapy is used to express nucleic acids for specific therapeutic purposes into a patient's cells. Is the introduction of a nucleic acid. That is, the nucleic acid itself is used as a drug. For example, Smart genes are delivered to patients with a recessive genetic disorder, such as cystic fibrosis. Can correct genetic defects and treat disease states. In other applications, Encodes toxins (e.g., diphtheria toxin, ricin, tk) to kill vesicles Gene delivery can be used, and other genes specific to kill infectious organisms Can be tailored. Other applications include incorporation of regulatory sequences near endogenous genes including. These different applications have many modes of delivery (e.g., in vitro, Many different target cells in vivo, in situ, intravenous, and germline modified About.   Gene therapy has the power of many large pharmaceutical manufacturers and some smaller Biotech companies into viable therapeutic approaches to treating human diseases Substantial financial and technical supply to develop gene therapy as a approach Led to focus the source. Although simple in theory, there are technical difficulties in gene therapy. There is no difficulty. Cell type as a target for the development of any gene therapy Identification of DNA, means for entry of DNA into these cells, Means for expressing useful levels of progeny products and hosts for gene therapy agents Requires shuffling of the immune response.   The requirements for any particular application depend on the vector to be developed and tested. Greatly influence the choice and profound effect. Possible variables in different applications are Gene transfer efficiency, gene expression efficiency, gene expression duration, Possibilities, including the ability to target appropriate cells and avoid inappropriate cells . A possible confounding predisposition is that the virus or delivery vehicle enters certain cells Or the inability to integrate into the chromosome of a particular cell, a transcriptional promoter Shutdown, loss of input DNA, destruction of treated cells, and Virus or gene product neutralization. All of these predispositions are viruses Choice of vector or non-viral delivery system, and the host Depends on the ability to respond to the system.   Most of the currently available components for constructing gene therapy vectors Have not evolved or been developed for therapy and therefore have many undesirable properties And may lack efficacy in the desired gene therapy application. For example, most Min eukaryotic virus optimizes virulence and virus reproduction Most non-viral DNA delivery systems have been developed for administration to humans. But not for experimental transfection in laboratory conditions. Designed to be.   The solution to the above difficulties and inefficiencies is that gene therapy has many of the Is necessary before it can be effective in the routine treatment of patients. The present invention addresses this need. And other needs, among others, used in gene therapy by recursive sequence recombination. By providing improved methods for improving vectors and other nucleic acids. Add                                Summary of the Invention   The present invention advances nucleic acids for use in gene therapy by recursive sequence recombination. Provide a way to The method involves the use of each other in at least two nucleotides. Recombining at least the first and second forms of the different segments To generate a library of recombinant segments. Then this library At least one recombinant segment from a gene for properties useful for gene therapy. Screened. Then, the lesser number identified by this screening Both one recombinant segments are the same as or different from the first and second forms Is recombined with a further form of the segment, and further Generate an library. This further library is then screened. This additional line has been developed for improvements in properties useful for gene therapy. At least one additional recombinant segment is identified from the library. Recombinant And additional cycles of screening, if necessary, Until it provides the desired level of properties useful for gene therapy Will be implemented.   In one embodiment, the invention relates to the use in gene therapy by recursive sequence recombination. Provide a method for modifying a nucleic acid segment for use, comprising: Steps: (1) At least first and first segments different at at least two positions Recombination of the two forms to produce a first set of recombinant segments: (2) Genetics Screening at least one recombinant segment for properties useful for offspring treatment (3) at least one recombinant cell generated by steps (1) and (2); Segment, the same as or different from the first or second form, Generating a second set of recombinant segments with the variant form of And (4) removing at least one recombination segment from the second recombination set Screening for properties useful for gene therapy. this In a further embodiment of the method, steps (1) to (4) are performed in which the Is repeated until the drug conferred properties useful for gene therapy. More of this method In embodiments, the nucleic acid segment can be a viral nucleic acid segment, or The viral nucleic acid segment may comprise a viral vector, or may comprise at least one Occurs in vivo or in vitro.   In one embodiment, the desired property to be obtained is at an improved virus titer. is there. Here, the recombinant segment is the growth of the virus on the cell for multiple generations. And screening as a component of the virus by isolation of progeny virus Progeny virus is a virus having a recombinant segment that confers improved titer properties Have been enriched.   In a second embodiment, the desired property is improved viral infectivity. Recombination Segment determines the percentage of the population of cells infected by the virus. It can be screened as a more viral component.   In a third embodiment, the desired property is an improved gene within the nucleic acid segment. Expression. The recombinant segment detects the expression of the recombinant segment in the cell Can be screened.   In a fourth embodiment, the desired property is improved or altered drug resistance. Recombinant segments involve exposing cells to drugs and selecting viable cells The surviving cells can be screened for improved or altered drug resistance. It is enriched for recombinant segments with properties.   In a fifth embodiment, the desired property is an improved or altered tissue specificity It is. Recombinant segments require the virus to be infectious by the virus A first population of cells to be infected and cells for which the nature of the infection by the virus is not desired Contacting a progeny virus from said first population of cells. By isolation, it can be screened as a component of the virus. Lus is a recombinant segment that confers infectious properties on a first subpopulation of the cells. Have been enriched.   In a sixth embodiment, the desired property is an improved viral capsid package. Aging ability. Recombinant segments allow the virus to grow on cells , And the progeny virus containing the recombinant segment by isolating the virus It can be screened as a component. Virus capsi containing recombinant segment The packaging capacity of the code increases during successive screening steps.   In a seventh embodiment, the desired property is episomal retention. Recombinant segment Cells containing the cells can be grown without selection for the recombinant segment. And then growing the cells with selection for the recombinant segment Cells that can be screened and survive selection will have improved episomal retention. It is enriched for cells that carry a recombinant segment with properties.   In an eighth embodiment, the desired property is that of the recombinant segment or its expression product. Of reduced immunogenicity. The recombinant segment is a mammalian segment And recovering the recombinant segments that survive after a predetermined period of time Can be screened.   In a ninth embodiment, the desired property is site-specific integration. Recombinant Segume Can be introduced into cells, and cells containing the desired integration site Screening can be performed by recovering a region of the DNA, which region is It is enriched for recombinant segments with heterologous integration properties.   In a tenth embodiment, the desired property is increased stability. Recombinant segment Subject the virus to destabilizing conditions and recover viable virus. And can be screened as components of the virus. Enriched for recombinant segments that confer the properties of   In an eleventh embodiment, the property is the ability to confer cellular resistance to a microbial infection. is there. Cells containing the recombinant segment are not capable of surviving infection by the microorganism. Can be screened.   In a twelfth embodiment, a method of the invention comprises introducing a non-viral form into a target cell. Evolve vector for The recombinant segment is a mammal Introduction into animals, this segment is integrated and expressed to Recovering cells from mammals that produce antisense or antisense RNA; and By recovering the recombinant segments from the cells.   In a thirteenth embodiment, the present invention provides a method for expression in a packaging cell line. Provides a method for improving the adeno-associated virus proteins rep and cap. This Cells containing the recombinant segments of these genes are flanked by terminal repeats (ITRs). Recombinant AAV (rAAV) containing marker genes Infect with par virus. Progeny rAAV and helpers produced by different cells Virus yield was determined and high relative yield of rAAV to helper virus Cells with a rate are selected.   In a fourteenth embodiment, the nucleic acid segment is a protein or an antisense R A coding sequence encoding NA, which is inserted into the genomic DNA of a mammalian cell. After integration of the segment.   In a fifteenth embodiment, the nucleic acid segment encodes a viral protein; In addition, the above-described properties allow the transfer of the cell line containing the nucleic acid segment into the cell line. Ability to package isolated viral DNA.   In a sixteenth embodiment, the nucleic acid segment encodes a DNA binding protein, The increased properties are dependent on the recipient of the vector encoding the DNA binding protein. Uptake by cells.   In a seventeenth embodiment, the invention is encoded by the nucleic acid sequence of SEQ ID NO: 1. With the amino acid sequence of SEQ ID NO: 2, the isolated recombinant as shown in FIG. O⁶-Methylguanine-DNA methyltransferase (MGMT) enzyme. This enzyme is present in the native human MGMT coding sequence, and Has at least one amino acid segment not present in the nucleotide sequence, and Present in the native non-human MGMT coding sequence and in the native human MGMT coding sequence May have at least one amino acid segment that is not present.   In another embodiment, the invention provides an adenovirus and phage f1 origin of replication. Phagemid-adenovirus capable of producing single-stranded DNA greater than 10 kilobases Provide irs.                             BRIEF DESCRIPTION OF THE FIGURES   Figure 1: In vitro shuffling of genes, a tool for “recursive sequence recombination”. It is a chime.   Figure 2: DNA binding conferring enhanced DNA uptake by recipient cells 3 is a scheme for selecting a protein.   Figure 3: To generate a recombinant form of MGMT using the recursive recombination method of the present invention. Oligonucleotide used for   Figure 4: Five known mammalian alkyltransferases-human, rat, ma 1 illustrates the natural diversity of mouse, hamster, and rabbit. Using this diversity To generate improved sequence diversity in the human MGMT gene.   Figure 5: Nucleotide of the improved human MGMT gene generated by the method of the present invention FIG. 3 illustrates the tide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2).   FIG. 6: illustrates the structure of the novel adenovirus-phagemid.                                   Definition   The term “screening” is generally a two-step process, where First, determine which cells express and do not express the screening marker. Then, those which physically separate cells having desired properties are described. The choice is Screenini where identification and physical separation are achieved simultaneously by expression of a selectable marker Is a form of signaling that, in some genetic situations, is a cell that expresses a marker. Survival the vesicles while killing other cells (or vice versa). Screw Luciferase, β-galactosidase, and green fluorescent markers Contains protein. Selectable markers include drug and toxin resistance genes. Self Although spontaneous selection can and does occur in the course of natural evolution, the method of the present invention provides Selection is performed by a human.   The term “exogenous DNA segment” is foreign or foreign to a cell, Alternatively, a host cell that is homologous to the cell but for which the element is not normally found A DNA segment located at a position within a nucleic acid.   The term "gene" broadens any segment of DNA that is associated with a biological function. Used to say. Thus, the genes may have a coding sequence and / or their Contains regulatory sequences required for expression. Genes can also be used, for example, for other proteins. Contains non-expressed DNA segments that form the recognition sequence.   The term "% sequence identity", "sequence identity", "sequence similarity" or "structural similarity" "Compares two sequences that are optimally aligned on a comparison window, both Determine the number of identical nucleobase positions occurring in the sequence and generate the number of matched positions Reduce the number of matched positions by the total number of positions in the comparison window Calculated or determined. Array alignment to align the comparison window Suitable alignment is Wisconsin Genetics Software Package Release 7.0, Gen Algorithm GAP in etics Computer Group, 575 Science Dr., Madison, WI , BESTFIT, FASTA and TFASTA Computerized Implementation Can be implemented.   The term "naturally occurring" is another term because it is artificially produced by humans. Used to describe controls that can be found naturally as being. For example, heaven Present in organisms (including viruses) that can be isolated from certain sources, and Polypeptide or polynucleotide not intentionally modified by a human in the laboratory Otide sequences occur naturally. In general, the term naturally occurring is representative of a species Refers to an object that is present in a non-pathogenic (disease-free) individual, such as is targeted.   The terms "isolated," "purified," or "biologically pure" The components usually associated therewith, substantially or essentially, as found in the natural state. Refers to substances that do not contain.   A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. Is tied. For example, a promoter or enhancer If it increases the transcription of a row, it is operably linked to the coding sequence. Ready for operation Linked means that the DNA sequences being linked are typically contiguous, and If the two protein coding regions need to be connected It means that it is a frame. However, in general, an enhancer is a promoter Works when separated by a few kilobases from, and the intron sequence is of variable length Some polynucleotide elements may be operable because Can be linked, but not continuous.   The specific binding affinity between two molecules, for example, between a ligand and a receptor, is It refers to the preferential binding of one molecule to another in a mixture of molecules. Minute The binding of the offspring has a binding affinity of about 1 × 10^FourM^-1~ About 1 × 10⁶M^-1Or larger Can be considered to be specific.                             Detailed description of the invention I. Introduction   The present invention is directed to obtaining improvements in properties or characteristics useful in gene therapy. Methods for evolving (ie, modifying) nucleic acids are provided. This modification or evolution The substrates for Different in each application. Candidate substrates for property acquisition or improvement in properties Examples include viral and non-viral vectors used in gene therapy. No. The method requires at least two variant forms of the starting substrate. Modified form Candidate substrates can exhibit substantial sequence or secondary structure similarity to each other, but They should also differ in at least two positions. The initial diversity between forms Can be the result of natural variations, for example, various variant forms (homologs) ) Can be obtained from various individuals or strains of the organism, including geographic variants, Constitute related sequences from the same organism (eg, allelic modification). is there Alternatively, an initial diversity can be induced, for example, the second variant form comprises the first variant Error-prone transcription (eg, error-prone PCR) or proofing Use of polymerase lacking reading activity (Liao (1990) Gene 88: 107-11 1) or a mutator strain (mutator host cells are (Discussed in more detail below) can be produced by replication of the first form . Initial diversity between substrates is greatly enhanced in subsequent recursive sequence recombination steps. It is.   The characteristics or characteristics that may be required to be acquired or improved vary widely, and Depends on the choice of substrate. For example, for viral and non-viral vector sequences The objectives of improvement are higher titer, more stable expression, and improved stability. , Higher specific target, higher frequency of integration, vector sequence or its expression Reduced immunogenicity of the product and higher expression of the gene product. Heredity Used to package viral vectors used in child therapy Genomic DNA from packaging cell lines Increasing the titer of the virus produced by the vesicle strain.   An improvement in or acquisition of a property is achieved by recursive sequence recombination . Recursive sequence recombination can occur in many different ways, as described in more detail below. It can be achieved in the order of formula and style. These modalities share some common principles. Minute To share. Recursive sequence recombination is a series of successive cycles to produce molecular diversity. Imposing recombination. That is, nucleic acid molecules that exhibit some sequence identity to one another. Produces millies, but differs in the presence of the mutation. In any given cycle, the set Transposition can occur in vivo or in vitro, intracellularly or extracellularly. further, The diversity arising from recombination can be mutated to either the substrate or the product for recombination. Applying conventional methods (eg, error-prone PCR or cassette mutagenesis) Can be enhanced in any cycle. In some cases, new Or improved properties or characteristics are from different individuals or strains of the organism Sequences of various variant forms, such as homologs, or identical When using related sequences from organisms, one round of in vivo or in vitro recombination It can be achieved after only a cycle.   Recombination cycles usually involve a small number of molecules with the desired properties or characteristics. At least one cycle of screening or selection follows. Recombination cycle in When performed in vitro, the products of recombination (ie, the recombinant segments) Is introduced into the cells prior to the screening step. The recombinant segment also Prior to screening, they may be ligated to a suitable vector or other regulatory sequence. Ah Alternatively, recombinant products produced in vitro may sometimes be obtained prior to screening. Packaged as a virus. When recombination is performed in vivo, Recombinant products can sometimes be screened in cells where recombination has occurred. other In the application of the recombinant segment is extracted from the cells and optionally Packaged as a virus before screening.   The nature of the screening or selection depends on the characteristics or characteristics to be The good will depend on the property or characteristic sought, and many examples are discussed below. The specific product of the recombination (recombinant segment) is thereby compared to the starting material. Understanding the molecular basis that has acquired new or improved properties or characteristics Usually not needed or desired. For example, gene therapy vectors have a variety of (Eg, coding sequences, regulatory sequences). Sequence, target sequence, stabilization sequence, and integration sequence). Each of these component arrays They can be changed and recombined simultaneously. Then, screening / selection For example, such improvements need to be attributed to any of the individual component sequences of the vector. Recombinant segments with increased expression stability in unneeded target cells Can be performed.   The first round of screening often involves high transfection efficiency and And in bacterial cells due to the ease of culture. Later times, those Recombinant segments for use in environments proximate to the intended environment of use Can be performed in mammalian cells to optimize At the end of screening Rounds can be performed on the correct cell type for the intended use (eg, stem cells). In some cases, the stem cells are, for example, immunogenic in the patient. Obtained from the patient to be treated for the purpose of minimizing the problem. In several ways In addition, the use of gene therapy vectors in treatment is itself a screening Used as times. In other words, persistent by the intended target cells of a patient Gene therapy vectors that are incorporated, integrated, and / or expressed in Is recovered from its target cells and used to treat another patient. You. Gene therapy vectors recovered from a patient's intended target cells have evolved (I.e., modified for specific uptake, integration and / or expression) Modified by recursive recombination towards improved or new properties or characteristics Enriched for the vector.   Screening or selection steps may be useful for gene therapy Identify Subpopulations of Recombinant Segments Evolved to Obtain the Desired Properties I do. Depending on the screen, the recombinant segments may be Component or free form. More than one screening Alternatively, a selection can be made after each round of recombination.   Screening / selection of at least one of the surviving recombinant segments, and Usually one harvest is exposed to a further round of recombination. These recombinant segs Can be either extrinsic segments that represent each other or the original substrate or additional It can be recombined with a variant. In addition, recombination can take place in vitro or in vivo. Can be A previous screening step required the desired recombinant segment as a component of the cell. If a component is identified, can the component be exposed to further recombination in vivo? , Or may be exposed to further recombination in vitro, or It can be isolated before performing the round. Conversely, the previous screening step was These components may be used to identify the desired recombinant segments as components of the virus or virus. Fragment can be introduced into cells to perform a round of in vivo recombination. How Regardless of whether it is performed, the second round of recombination is performed on the recombinant segment resulting from the previous round. Additional recombinant segments containing more diversity than exist in the Produce.   The second round of recombination is screened according to the principles described above for the first round / Further rounds of selection may follow. The stringency of screening / selection is Can be increased between times. In addition, the nature of the screening and the screening The property to be used is if an improvement in more than one property is desired, or It may change from time to time if it is desired to acquire many new properties. Then In a further round of recombination and screening, the recombinant segment is Done until it has evolved sufficiently to obtain a regular or improved property or function Can be II.Format for recursive sequence recombination   Illustrative formats and examples for using recursive sequence recombination are, at times, DNA shuffling. Called fling, sexual PCR or molecular bleeding, March 25, 1996 Co-pending application filed with United States Application No. (USSN) 08 / 621,859, Agent Control Number 16 528A-014612; International published as WO 95/22625, filed February 17, 1995 Application PCT / US95 / 02126; Stemmer (1995) Science 270: 1510; Stemmer (1995) Gene 164: 49-53; Stemmer (1995) Bio / Technology 13: 549-553; Stemmer (1994) Proc. N atl.Acad.Sci.USA 91: 10747-10751; Stemmer (1994) Nature 370: 389-391; Cramer i (1996) Nature Medicine 2: 1-3; Crameri (1996) Nature Biotechnology 14:31 5-319 describes the present inventors and co-workers.(1) In vitro format One embodiment for shuffling DNA sequences in vitro is shown in FIG. It is. The first substrate for recombination is the sequence involved (eg, from an individual or a variety of organisms). Various variant forms such as homologs from strains, or allelic variants , Related sequences from the same organism). In FIG. 1, panel A, X Indicates the site where The sequence can be DNA or RNA and is either recombined or Depending on the size of the gene or DNA fragment to be reconstructed Can get. Preferably, the sequence is between 50 bp and 50 kb.   The pool of related substrates is about 5 bp to 5 bp, as shown in FIG. Converted to overlapping fragments of kb or more. Often, for example, The size of the fragments is about 10 bp to 1000 bp, and sometimes DNA fragment The size of the target is about 100 bp to 500 bp. This conversion can be done in a number of different ways (eg, , DNase I or RNAse digestion, random shear, or partial restriction enzyme digestion). Can be brought. Consideration of protocols for nucleic acid isolation, manipulation, enzyme treatment, etc. For example, see Sambrook, Molecular Cloning: A LABORATORY MANUAL (second edition) , 1-3, Cold Spring Harbor Laboratory, (1989) (Sambrook), and CURRENT  PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, Greene Publishing and Wiley- See Interscience, New York (1987) (Ausubel). Specific length and distribution The concentration of nucleic acid fragments in a row is often less than 0.1% or 1% of the total nucleic acid weight. Few. The number of different specific nucleic acid fragments is usually at least about 100, 500, Or 1000.   Mixed populations of nucleic acid fragments can be prepared using a variety of techniques (eg, heating, chemical denaturation, DNA At least partially single-stranded nucleic acid forms (including use of binding proteins, etc.) Is converted to The conversion is performed at about 80 ° C. to 100 ° C. to form a single-stranded nucleic acid fragment. C., more preferably to 90-96.degree. C., and then annealing again. Can be brought about. The conversion also involves single-stranded DNA binding protein (Wold (1997) A nnu.Rev.Biochem. 66: 61-92) or the recA protein (Kiianitsa ( 1997) Proc. Natl. Acad. Sci. USA 94: 7837-7840). Can be brought. Then has sequence identity with other single-stranded nucleic acid fragments The single-stranded nucleic acid fragment is cooled to 20 ° C to 75 ° C and preferably to 40 ° C to 65 ° C. Can be annealed again. Regeneration is polyethylene glycol (PEG), other volume exclusion reagents or salts can be accelerated. The salt concentration is Preferably, the salt concentration is between 0 mM and 200 mM, more preferably the salt concentration is between 10 mM and 100 mM. salt Can be KCl or NaCl. The concentration of PEG is preferably between 0% and 20%, More preferably, it is 5% to 10%. The fragment that anneals again is shown in FIG. As shown in panel C, it can be from a different substrate. Annealed nucleic acids Fragments are made of nucleic acid polymerases (eg, Taq or Klenow) and dNTPs (eg, That is, it is incubated in the presence of dATP, dCTP, dGTP, and dTTP). If the region of sequence identity is large, Taq polymerase should be annealed between 45-65 ° C. Can be used at the operating temperature. If the region of identity is small, Klenow polymerase An annealing temperature between 20 and 30 ° C can be used. Polymerase, Anilin Random nucleic acid fragments before, simultaneously with, or after annealing To the cement.   To produce a collection of polynucleotides containing fragments of different permutations Denaturation of overlapping fragments in the presence of polymerase The production and incubation processes sometimes involve the shaking of nucleic acids in vitro. It is called ruffling. This cycle is repeated a desired number of times. Preferably The cycle is repeated 2 to 100 times, more preferably the sequence is repeated 10 to 40 times It is. The resulting nucleic acid is preferably between about 50 bp and about 100 kb, as shown in FIG. Or a family of 500 bp to 50 kb double-stranded polynucleotides. The group is out Represents a variant that shows a similar identity to the substrate, but also differs at some positions. You. The population has more members than the starting substrate. Arising from shuffling The population of fragments is cloned into a vector, if necessary, and Used to transform cells.   In one embodiment utilizing in vitro shuffling, the use of recombinant substrates If the run is a substantial fraction, typically incompletely stretched by at least 20% or more Can be produced by amplifying the full-length sequence under conditions that produce a longer amplification product. You. Another embodiment uses template DNA to produce less than full-length amplification products. Use primers to prime. Amplification product (this is an incompletely expanded amplification Product) is denatured and at least one additional reannealing and It is subjected to a cycle of amplification. At least one re-annealing and amplification This modification, which provides a substantial fraction of incomplete extension products, is referred to as (Stuttering) ". Partial extension in subsequent amplification rounds The product (less than full length) will anneal again and on different sequence related template species Prime extension. In another embodiment, the conversion of the substrate to a fragment comprises: It can result from partial PCR amplification of the substrate.   In another embodiment, the mixture of fragments comprises one or more oligonucleotides. Spiked with Oligonucleotides were pre-characterized of the wild-type sequence It can be designed to include sites for mutation, or natural modification between individuals or species. Oligonucleotides also allow annealing with wild-type fragments Includes sufficient sequence or structural homology to flank such mutations or modifications. Ani The cooling temperature can be adjusted according to the length of homology.   In a further embodiment, the recombination comprises template switching (eg, DNA fragments from one template are related but different templates At least in one cycle) Occur in Template switching is performed using recA (Kiianitsa ( 1997) supra) rad51 (Namsaraev (1997) Mol. Cell. Biol. 17: 5359-536). 8), rad55 (see Clever (1997) EMBO J. 16: 2535-2544), r ad57 (see Sung (1997) Genes Dev. 11: 1111-1121) or other polymer (E.g., viral polymerase, reverse transcriptase) . Template switching also involves increasing DNA template concentration Thus it can be increased.   Another embodiment utilizes at least one cycle of amplification. This is related Using a sequence and a collection of overlapping single-stranded DNA fragments of different lengths Can be done. The fragment was a single-stranded DNA phage (M13 (Wang (1997) Biochemis). try 36: 9486-9492))). Each fragment Hybridized to a second fragment from the collection, and Priming the pseudostrand extension, thus forming a sequence-recombinant polynucleotide. In a further modification, the variable length ssDNA fragment comprises a first DNA template. Pfu, Taq, Vent, Deep Vent, U1Tma DNA polymerase or other DNA Can be made from a single primer by the enzyme (Cline (1996) Nucleic Acids  Res. 24: 3546-3551). The single-stranded DNA fragment is the second Kunkel type Used as a primer for the template. This is a uracil-containing ring Consists of ssDNA. This is the multiple of the first template into the second template. Causes a substitution. Levichkin (1995), Mol. Biology, 29, 572-577; Jung (1992) Gen See e 121: 17-24.Reintroducing shuffled genes into cells in vitro   In a further embodiment, the whole cell and organism is an in vitro shuffle. Regression cycle evolves the transgene in its cells and organisms Can be improved. The transferred gene is exposed to the recurrent recombination method of the present invention. And the shuffled sequence library is returned to the cell / organism for selection. It is. If multiple copies of the modified transgene are reintegrated into the cell, This method is useful, while in a preferred modification of this selection assay, A copy-only modified transgene is inserted into each cell. Another of this selection assay Preferred modifications include reducing the expression variability of transcription of the modified transgene. This can result from differences in the chromosomal location of the integration site. It is prescribed It also requires a means for site-specific integration of the modified transgene. These people The method uses an episomal vector that can integrate site-specifically into the chromosome Can be used to evolve   Use of a retrovirus to return a transgene modified for selection to cells Have the advantage that they integrate as a single copy. But this insertion , Not site-specific, that is, retroviruses insert. Adenovirus and ars plasmids also contain modified transgenes , But they integrate as multiple copies. Wild-type AAV integrates into chromosome q19 as a single copy, but is commonly used No modified AAV is performed. Homologous recombination can also be a modified recombinant segment (Transgene) to be inserted into the chromosome, but this method is not Yes, and can result in two copies of integration into a set of chromosomes. Eliminate these problems In order to achieve this, one embodiment of the present invention provides for the transfer of genes to specific constant locations in the genome. Utilizes a site-specific integration system to target offspring. A preferred embodiment is Cre / LoxP Alternatively, use the relevant FLP / FRT site-specific integration system. Cre / LoxP system is Cre Rico Using site specific insertion and identification called "LoxP site" using the enzyme Excision of viral or phage vector into the palindrome 34 base pair sequence Mediate. A LoxP site is inserted into the mammalian genome of choice, for example, in a homology set. Permutation (see Rohlmann (1996) Nature Biotech. 14: 1562-1565). A transgenic animal containing a LoxP site can be generated. Genome is in desired position Possesses the Cre recombinase gene when manipulated to include a LoxP site Infection of such cells with the transfected vector is performed by Efficient site-specific integration into DNA. This approach replicates between cycles Possible and modified transgenes at certain, defined locations (recombinant Sequence). Therefore, the transgene of interest is Can be modified in vitro using sequence recombination techniques and with minimal noise In vivo / in situ selection for new or improved properties in an optimal way Can be re-inserted into the cell. This technique is also used as discussed below. Can be used in vivo. For example, Agah (1997) J. Clin. Invest. 100: 169-179; Akagi (1997) Nucleic Acids Res. 25: 1766-1773; Xiao (1997) Nucleic Acids Res 25 : 2985-2991; Jiang (1997) Curr Biol 7: 321-R323, Rohlmann (1996) Nature Bio tech. 14: 1562-1565; Siegal (1996) Genetics 144: 715-726; Wild (1996) Gene 1 See 79: 181-188. The evolution of Cre is discussed in further detail below.(2) In vivo format (a) Plasmid-plasmid recombination   The recursive recombination method of the present invention involves plasmid-plasmid recombination. Book In embodiments and other embodiments, the first substrate for recombination is a nucleic acid of interest (eg, For example, genes, vectors, transcription regulatory sequences, etc.) A collection of pride. Mutant bears have substantial sequence identity to each other; eg, between substrates Have sufficient sequence identity to allow homologous recombination at (Datta (1997) Proc. Natl. Acad. Sci. USA 94: 9757-9762; Shimizu (1997) J. Am. Mol. Biol. 266: 297-30 5; Watt (1985) Proc. Natl. Acad. Sci. USA 82: 4768-4772). Poly The diversity between the nucleotides may be natural (e.g., allelic or species variant), induced, Derived (e.g., produced in vitro, such as by error-prone PCR) , Light (1995) Bioorg. Med. Chem. 3: 955-957), or in vitro It is the result of the recombination. Diversity also involves alternative and / or mixed codon usage It can result from the resynthesis of the gene encoding the native protein. Between substrates Both have sufficient diversity that recombination can produce more diverse products than the starting material. Must be present. At least two substrates differing in at least two positions Must exist. However, in another embodiment, 10^Three~Ten⁸Member substrate Libraries are used. The degree of diversity depends on the length of the substrate being recombined And the extent of the functional changes to be evolved. In the position between 0.1-50% Diversity is typical.   A variety of starting substrates or recombinant segments modified by the methods of the present invention It can be incorporated into rathmids. In one embodiment, the plasmid is a standard clone. A vector (eg, a bacterial multicopy plasmid). However, In another embodiment described below, the plasmid comprises a recruitment function. Starting substrate or The recombinant segments can be incorporated into the same or different plasmids. For a while For example, at least two different types of plasmids with different types of selectable markers Sumid allows selection of cells containing at least two types of vectors Used for Also, if different types of plasmids are used, different Rasmids were obtained from two separate mismatched groups and were given two different intracellular It may allow stable coexistence of Rasmid. Nevertheless, the same incompatible group If the original plasmid coexists in the same cell for a time sufficient to cause homologous recombination, obtain.   Plasmids containing a variety of substrates may be first prepared by any transfection method (e.g., Chemical transformation, natural capacity, electroporation, viral transduction Introduced into prokaryotic or eukaryotic cells (e.g., See, for example, Sambrook for a detailed description of introducing DNA into cells; Hap ala (1997) Crit. Rev. Biotechnol. 17: 105-122). Often, plasmids At or near the sum concentration (for maximum transfection capacity) Increases the probability that more than one plasmid will enter the same cell. Various substrates Alternatively, the plasmid containing the recombinant segment may be transferred simultaneously or multiple times. Can be controlled. For example, in the latter approach, the cell is the first of the plasmid. Transfectants are selected and transfected. And then infected with a second aliquot of the plasmid.   When the plasmid is introduced into the cell, the recombinant gene or other nucleic acid segment Recombination between the substrates to produce Occurs in cells containing multiple different plasmids. But only one Cells that have received only one plasmid cannot participate in recombination, and Plasmids are propagated in mutator cells (described below) or Otherwise, if point mutations accumulate (ie, Substrates for such plasmids that can contribute to sequence diversity but for evolution The potential contribution (sequence modification) is not fully available. Rate of evolution (ie, book Modification of the nucleic acid sequence by the method of the invention) involves involving all substrates in recombination. Thus, it can be increased. In one embodiment, this is the transfected This is achieved by subjecting the cells to electroporation. Electropo The conditions for the translation are those conventionally used to introduce foreign DNA into cells. (Eg, 1,000-2,500 volts, 400 μF, and 1-2 mM gap). This Under these conditions, the plasmid is exchanged between cells, thereby assembling all substrates. It becomes possible to be involved in exchange. In addition, the products of the recombinants Further rounds of recombination with the substrate may be performed.   In another embodiment, the rate of evolution (ie, the rate of recursive sequence modification) is also It can be augmented by the use of a conjugative transfar. Zygotic transition To utilize, the substrate is cloned into a plasmid carrying the MOB gene, And the tra gene is also provided in cis or trans to the MOB gene. The effect of conjugative transfer allows the plasmid to move between cells and Allows recombination between any substrates, and the products of previous recombination simply increase the culture. Very similar to electroporation in that it occurs by propagation are doing. Learn more about how conjugative transfer is exploited in these vectors Details are discussed in more detail below (see, eg, Cabezon (1997) Mol. Gen. Genet. 254: 400-406).   Rate of evolution also fuses cells to induce plasmid or chromosome exchange Can be increased. The fusion can be a chemical agent (eg, PEG), or a virus. Virus or viral proteins (eg, influenza virus hemagglutinin , HSV-1 gB and gD, or fusogenic liposome (See Dzau (1996) Proc. Natl. Acad. Sci. USA 93: 11421-11425). See).   The rate of evolution can also be determined by mutator host cells (eg, bacteria Mut L, S, D, T H mutator cells, insect (Drosophila) and mouse mutator cells, And human cell lines with defective DNA repair mechanisms (e.g., telangiectasia ataxia (ata xia telengiectasia) (cells from patients) (Morgan (199 7) Cancer Res. 57: 3386-3389; Greener (1997) Mol. Biotechnol. 7: 189-195; Maso n (1997) Genetics 146: 1381-1397; Aronshtam (1996) Nucleic. Acids Res 24: 2498- 2504; Seong (1995) Int. J. Radiat. Oncol. Biol. Phys. 33: 869-874; Wu (1994) J. Bacteriol. 176: 5393-5400; Rewinski (1987) Nucleic. Acids Res. 15: 8205-8 215; Aizawa (1986) Jpn. J. Cancer Res. 77: 327-329).   The time at which cells grow and recombination occurs, of course, depends on the cell type Can vary, but is generally not critical. Because even a small degree of recombination Because it can substantially increase the diversity compared to the starting material. Recombinant gene A cell having a plasmid containing is screened for a desired function or Served for selection. For example, if the evolving substrate contains a drug resistance gene, Select for resistance. Cells that survive screening or selection should be Can be subjected to the above screening / selection, subsequent recombination, or an additional number of It can be directly subjected to recombination.   The next round of recombination may take several different formats depending on the previous round. Can be achieved. For example, in one embodiment, a further round of recombination is simply Resume electroporation or conjugation-mediated cell-to-cell transfer of the plasmid ( Repetition). In another embodiment, the fresh substrate (previous substrate (Same as or different from) transfect cells surviving selection / screening Can be effected. New substrates can be used for plasmids with different selective (selection) markers. From an incompatible group that differs from the sumid vector and / or the original plasmid In a plasmid vector. Selectable marker can be selected for transformed cells Phenotype. For example, markers may be antibiotic resistance, especially chloramphenic , Kanamycin, G418, bleomycin and hygromycin To allow selection of those cells that have been transformed with the desired DNA sequence. . See, for example, Blondelet-Rouault (1997) Gene 190: 315-317. Neo Mai A selectable marker that confers resistance to a substrate such as syn or hygromycin Since the gene can only be used in tissue culture, chemical resistance genes are also It can be used as a selectable marker in vitro and in vivo. Various target details Vesicles are P-glycoprotein, multiple drug resistance-related protein transporter, dihydrogen Lofolate reductase, glutathione-S-transferase, 06-alkylguani DNA alkyltransferase (Tano (1997) J. Biol. Chem. 272: 13250-13254) ) Or aldehyde reductase (Licht (1997) Stem Cells 15: 104-111) Is made resistant to an anticancer drug by transfer of a chemical resistance gene encoding   In a further embodiment, the cells surviving the selection / screening are 2 Can be subdivided into two subpopulations and plasmid DNA from one subpopulation Transfected. Here, plasmid-derived groups from the two subpopulations The quality undergoes further rounds of recombination. In either of the latter two embodiments, Evolution rate can be DNA extraction, electroporation, conjugation, or mutator -Can be increased by the use of any of the above techniques involving cells. Still further In embodiments, DNA from cells surviving the screening / selection is extracted. And can be subjected to in vitro DNA shuffling.   After the second round of recombination, additional rounds of screening / selection may be performed. One In certain embodiments, the screening or selection increases stringency. The reaction is performed under the following conditions. If desired, additional rounds of recombination and selection / screening Can be performed using the same strategy used in the second round. Recombinant And using the next round of selection / screening, the surviving recombinant substrate Evolve to obtain the desired phenotype or trait. Typically, this of the present invention Recombination that has achieved the desired phenotype in recombination methods and other recursive recombination methods The final product may differ from the starting (starting) material by 0.1% to 25%. The present invention Method is based on the calculated ratio of naturally acquired mutations (10 per generation).^-9position About one mutation per Anderson, (1996) Proc. Natl. Acad. Sci. USA 93,906 (See -907) Larger percentage of magnitude (e.g., at least 10 times, 100 (1000-fold, 1000-fold or 10,000-fold) nucleic acid sequences can be evolved / altered.   (b) Virus-plasmid recombination   The recursive recombination method of the invention involves virus-plasmid recombination. Plastic The strategy used for sumid-plasmid recombination is also viral-plus In another embodiment of the present invention, which includes mid recombination or phage-plasmid recombination. Can be used in However, some additional, especially regarding the use of viruses The annotation is appropriate. The first substrates for recombination are plasmid and viral vectors. Cloned into both Which substrate is inserted into the viral vector, And which substrate is inserted into the plasmid is usually not important, Usually, viral vectors will contain different substrates from the plasmid. Less than As before, plasmids (and viruses) typically contain a selectable marker. No. Both plasmid and viral vectors are transfected as described above. Can be introduced into the cells by the application. However, a more efficient procedure is to use a plasmid Transfect cells, select transfectants, and To infect transfectants. Efficiency of infection of many viruses Is approximately equal to 100% of the cells, Most cells infected with plasmids and plasmids with different substrates Including both.   Homologous recombination occurs between the plasmid and the virus, And recombinant virus. Intracellular DNA in double- and single-stranded form For some viruses, such as filament-like phages, that are present on both Can both be involved in recombination. Viruses must kill cells quickly If recombination is increased by the use of electroporation or conjugation, Plasmids can be transferred between cells. Recombination can also be performed by progeny from one cell. By allowing the virus to reinfect other cells, Types can be increased. For some virus types, virus Infected cells show resistance to superinfection. However, such resistance is not Infections and / or alterations in viruses that have reduced resistance to superinfection It can be overcome with a heterologous strain.   The result of infecting plasmid-containing cells with a virus depends on the nature of the virus. You. Some viruses (e.g., filamentous phage) are positive in cells. Stably with the amide, and also extrude progeny phage from the cells (Russel (1997) Gene 192: 23-32.). Other viruses (e.g., cosmidgeno (Lambda) with a cell, as in a plasmid, without producing progeny virions. It is stable inside. Other viruses (eg, T phage and soluble λ) Undergoes recombination with the rasmid, but ultimately kills the host cell and the plasmid DN Destroy A. For viruses that infect cells without killing the host, recombinant Cells containing plasmids and viruses can be used as in plasmid-plasmid recombination Can be screened / selected using this approach. Selection / Screening Progeny virus extruded by cells that survived is also recovered and paired. It can be used as a substrate for the next round of replacement. For viruses that kill host cells, The recombinant gene obtained from the recombination remains only in the progeny virus. screening Or, if the selection assay requires expression of the recombinant gene in cells, Genes are transferred from the progeny virus to another vector (e.g., a plasmid vector). And transfect cells before selection / screening It must be.   For filamentous phage, the product of the recombination has survived the recombination Cells and phages extruded from these cells. Recombinant The dual source of product is several compared to plasmid-plasmid recombination. To provide further options. In one embodiment, the DNA is in vitro recombinant. Can be isolated from phage particles for use in a single round. In another embodiment, the offspring The phage is the cells or cells that survived the previous round of screening / recombination. Fresh cells transfected with fresh substrate for Can be used to infect or infect.   (c) Virus-virus recombination   The recursive recombination method of the invention also includes virus-virus recombination. Step Rasmid-plasmid recombination and plasmid-virus recombination are described. Principles can be applied to virus-virus recombination with some modifications . The first substrate for recombination is cloned into a viral vector. Like In a preferred embodiment, the same vector is used for all substrates. Preferably A virus is a virus that does not kill cells, either naturally or as a result of mutation. Insert Later, some viral genomes can be packaged in vitro. Package The virus is used to infect cells at high multiplicity, Are more likely to accept multiple viruses carrying different substrates.   After the initial infection, the next steps depend on the nature of the infection as discussed in the previous section. For example, if the virus is a λ cosmid or M13, Fl or Fd phagemid If you have a phagemid genome, the phagemid will appear intracellularly like a plasmid. And undergo recombination simply by growing the cells. Recombination is It is particularly efficient among single-stranded forms of intracellular DNA. Recombination of the cell It can be increased by troporation. Recombination following selection / screening Cosmids containing the gene from the surviving cells (e.g., cos-lysogenic host Harvested (by heat induction of cells), repackaged in vitro, and further Can be used to infect fresh cells at high multiplicity for any round of recombination .   If the virus is a filamentous phage, a set of replicating forms of DNA Transposition occurs by growing a culture of infected cells. Select / Screen Virus vector having recombinant gene with improved properties by leaning Cell colonies containing phage were identified along with phage extruded from such cells Is done. The next option is essentially the same as plasmid-viral recombination.   (d) Chromosome-plasmid recombination   The recursive recombination method of the present invention also includes chromosome-plasmid recombination. This Format evolves both chromosome and plasmid-carrying substrates Can be used for In this format, many chromosomal genes contribute to the phenotype Or where the exact location of the chromosomal gene to evolve is unknown. Especially useful. The first substrate for recombination is cloned into a plasmid vector. Be tuned. If the chromosomal gene to be evolved is known, the substrate A family of sequences that show sequence identity but show some diversity with chromosomal genes Is composed. If the chromosomal gene to be evolved is not located, the first substrate Usually show no sequence identity to genes that should be evolved in only a small number. A library of different DNA segments. Plasmid carrying substrate and chromosome inheritance Diversity between offspring can be induced by mutagenesis, as described above, or By obtaining a plasmid-carrying substrate from a different species than the chromosome-carrying cell species Can be induced.   Plasmids carrying substrates for recombination can be used to create new or modified traits Transfect cells with chromosomal genes that are evolved / altered to acquire Is done. Evolution by recursive recombination occurs simply by growing the culture. Can get. In another embodiment, the nucleic acid sequence modification is conjugation or electroporation. Can be accelerated by transferring the plasmid from one cell to another. Sarana In some embodiments, evolution by recursive recombination is performed using mutator host cells. Thus, or a non-mutator evolving with a mutator host cell The host cells are seeded and the plasmid is plated by electroporation or conjugation. It can be further accelerated by inducing intracellular metastasis. Preferably for sowing The mutator host cells used are purely evolving non-mutator cells. A negative selectable marker is included to facilitate the isolation of a successful culture. Choice / Screening to obtain or modify desired properties or functions Cells carrying the evolved chromosomes and / or plasmids are identified.   The next round of recombination and selection / screening was performed with plasmid-plasmid recombination. And proceed in a similar manner to that described above. For example, further recombination Recombination in combination with plasmid electroporation or conjugation transfer By growing the surviving cells. Alternatively, recombination A plasmid carrying additional substrate for is introduced into surviving cells obtain. Preferably, such plasmids are from different mismatched groups and Carrying a selectable marker different from the original plasmid and at least two different Allows selection of cells containing rasmid. As a further alternative, plasmids and And / or chromosomal DNA is isolated from a subpopulation of surviving cells, and A second subpopulation can be transfected. Chromosomal DNA is transfected Prior to cloning, it can be cloned into a plasmid vector.   (e) Virus-chromosome recombination   The recursive recombination method of the invention also includes chromosome-viral recombination. First As in the described embodiment, the virus usually does not kill cells. And often phage or phagemid. Procedure is substantial This is the same as plasmid-chromosome recombination. Substrate for recombination is in the vector Cloned. The vector containing the substrate is then transfected into the cells. Or packaged in vitro and transferred to cells by infection Can be entered. The viral genome is used to grow host cultures simply by growing the culture. Replaces with color body. Evolution is based on electroporation of viral genome into cells By metastasis in cells or by reinfection of cells with progeny virions Can be accelerated. Screening / selection allows new or modified features Chromosomes and / or viral genes that have evolved to acquire sex or a desired function Nome-bearing cells are identified.   There are several options for the next round of recombination. For example, Nom is selected by electroporation / between cells surviving recombination Can be moved. Alternatively, push selection / screening from surviving cells. The released viruses are pooled and used to superinfect cells at high multiplicity. Can be used for Alternatively, a fresh substrate for recombination is Any of the ils vectors can be introduced into cells.III . Vectors used in gene therapy   The present invention provides vectors by recursive recombination for use in gene therapy. A method for modifying is provided. Broadly, gene therapy vectors are Exogenous polynucleotides produce medically useful phenotypic effects on mammalian cells It is. Vectors may or may not have an origin of replication. For example, to patients For propagation of the vector prior to administration, it is useful to include an origin of replication in the vector. is there. However, the origin of replication does not allow the vector to integrate into the host chromosomal DNA or When designed to bind to primary mRNA or DNA, they are often removed before administration. obtain. Vectors used in gene therapy can be viral or non-viral possible. Viral vectors are usually introduced into patients as components of the virus . Exemplary Vector Incorporating a Nucleic Acid Modified by the Recursive Recombination Method of the Invention Are, for example, vectors based on adenovirus (Cantwell (1996) Blood 88: 4676- 4683; Ohashi (1997) Proc Natl Acad Svi USA 94: 1287-1292), Epstein Bar Virus-based vectors (Mazda (1997) J Immunol Methods 203: 143-151) , Adenovirus-associated virus vector, Sindbis virus vector (Strong (1997) Gene Ther 4: 624-627), herpes simplex virus vector (Kennedy (199 7) Brain 120-1245-1259) and retroviral vectors (Schubert (1997) Curr Eye Res 16: 656-662).   Can non-viral vectors (typically dsDNA) be transferred as naked DNA? Or transfer enhancing vehicles (eg, receptor recognition proteins, liposomes, Lipoamines, or cationic lipids). This DNA is Cells can be transferred using a variety of well-known techniques. For example, naked DNA interacts with the cell membrane Through the use of liposomes that fuse or undergo phagocytosis (ie, Attached to liposomes, which bind to cell surface membrane protein receptors that generate (Or by using a ligand attached or directly attached to DNA) . Alternatively, the cell enhances transport of DNA into the cell without damaging the host cell To For transmission. Those skilled in the art will recognize that DNA transfer is known to transport DNA into cells. Synthetic proteins (eg, HBGF-1) may be used. To deliver naked DNA to cells These procedures are useful in vivo. For example, using liposomes More so, especially if the liposome surface has a ligand specific for the target cell. If otherwise preferentially directed to a particular organ, those skilled in the art It may provide for the introduction of DNA into cells / organs.   A. Virus-based method   Various viral vectors (eg, retrovirus, adenovirus, adenovirus) Associated virus and herpes virus) are used in gene therapy. So They often contain two components, the modified viral genome and the envelope structure surrounding it. (Generally, Smith (1995) Annu. Rev. Microblol. 49, 807-838) However, sometimes viral vectors can be introduced in a naked form, or Coated with proteins other than the ils protein. Most recent vectors One has a coat structure similar to the wild-type virus. This structure holds the viral nucleic acid It is packaged and protected, and provides a means for binding and invading target cells. Only However, viral nucleic acids in vectors designed for gene therapy can vary widely. Can be The goal of these changes is to use available packaging cells or helpers. Virus in target cells while maintaining the ability to grow in vector form in cells Dissemination of the exogenous DNA sequence into the viral genome Providing a space and incorporating new sequences encoding the gene of interest. To enable proper expression of Therefore, a vector nucleic acid generally comprises two Contains: Essential cis Action for Replication and Packaging in Helper Strains Transcription units for viral sequences and exogenous genes. Other virus functions specific Expressed in trans in a packaging or helper cell line.     (1) Retro virus   Retroviruses are large classes of packages that contain single-stranded RNA as the viral genome. Including membrane viruses. During the normal viral life cycle, viral RNA is reverse transcribed, The resulting double-stranded DNA integrates into the host genome and is expressed over time. As a result, infected cells can continuously transmit the virus without apparent harm to the host cells. To scrap. The viral genome is small (about 10kb) and its prototype organization Is extremely simple, gag, group-specific antigen, or core protein; pol, reverse transcription Enzymes; and env, three genes encoding viral envelope proteins including. The end of the RNA genome is called the long terminal repeat (LTR), and the promoter And sequences involved in enhancer activity and integration. The genome also Required for packaging viral RNA for production of separate envelope mRNA Sequences and splice acceptor and donor sites. Most leto Virus can only integrate into replicating cells, whereas human immunodeficiency virus (HIV) Seems to be an exception. This property makes it a retrograde vector for gene therapy. It may limit the use of the virus.   Retroviral vectors are relatively simple, 5 'and 3' LTR, packaged And a transcription unit composed of the gene of interest (this is typically the expression Is a set). In order to propagate such vectors, so-called package Deleting virus function must be provided in trans using a soaking cell line . Such cells contain integrated copies of gag, pol and env, Parvirus sequences also lack packaging signals so that they are not encapsidated Is operated as follows. Added to or added to vector and packaging cells Additional features removed from this may make the vector more effective or helper -Reflects attempts to reduce the likelihood of coexistence by the virus.   The main advantage of retroviral vectors is that they integrate into the chromosome and thus It is possible to enable long-term expression in some cases. They are grown in relatively large amounts But requires care to ensure that no helper virus is present It is.     (2) Adenovirus   Adenoviruses include a large class of non-enveloped viruses containing linear double-stranded DNA. No. The normal life cycle of the virus does not require cell division, but imposes on permissive cells. Productive infections during which large amounts of virus accumulate. Productive infection cycle Takes about 32-36 hours in cell culture and is biphasic (early phase Before and during the late phase, the synthesis of structural proteins and viral DNA While being built on the lion). In general, adenovirus infection affects humans Associated with mild illness in   Adenovirus vectors are somewhat larger than retrovirus or AAV vectors And more complex. This is, in part, a small fraction of the viral genome. Only the case is taken from most current vectors. More inheritance If the children are removed, they are provided in trans to generate the vector. It is. This has hitherto proved difficult. Instead, adeno Two general types of virus-based vectors have been studied (E3 deletion vectors). Vector and E1 deletion vector). Some worms in wild-type experimental stock Ruth lacks the E3 region and can grow in the absence of helpers. This ability is available at E3 It does not mean that the gene product is not required in the wild type, but simply It means that replication in feeder cells does not require them. Deletion of E3 region Is the insertion and insertion of exogenous DNA sequences to generate a vector that allows productive infection. And the transient synthesis of relatively large amounts of the encoded protein.   Deletion of the E1 region disables adenovirus, but such vectors are still Can be propagated. It contains the E1 region of Ad5 and constitutively expresses the E1 protein This is because there are established established human cell lines (referred to as “293”). Adenovirus Most modern gene therapy applications, including E1 Using a replacement vector.   The main advantage of adenovirus vectors is that they can be used in a wide range of cells and tissues. And efficient episomal gene transfer in Is to multiply in quantity. The main drawback is that the host response to the virus Appear to limit the ability to repeat Requires a progeny vector.   In another embodiment, the recursive recombination method of the invention comprises a DNA having a size greater than 10 kb. Construct a novel adenovirus-phagemid capable of packaging inserts Used for The origin of the phage f1 origin in the plasmid using the method of the invention Incorporation also involves the entire genome of the virus (eg, the 36 kb fragment of human adenovirus). Create a new in vivo shuffling format that can evolve . The widely used human adenovirus type 5 (Ad5) has a genome size of 36 kb Having. This large genome is transformed in vitro with a high percentage of non-viable recombinant variants. Shuffling without causing an excessive number of changes that can cause It is difficult. Minimize this problem and achieve Ad5 whole-genome shuffling To this end, an adenovirus-phagemid was constructed. Ad-phage mi of the present invention Accepts inserts as large as 15 kb and 24 kb, and ssDN of that size It was shown to produce A effectively. In a further embodiment, a larger DNA insert Pieces (about 50-100 kb in size) are inserted into the Ad-phagemid of the invention; To generate a full-length ssDNA corresponding to a large insert. Such a big ssDNA flag The generation of a fragment evolves the entire viral genome (ie, the recursive set of the present invention). Means for modifying by a replacement method). Therefore, the present invention Pieces (> 10 KB) can be cloned and corresponding to their large insert, ss First unique phagemid system capable of producing DNA in vitro and in vivo provide.   The genome of the relevant serotype of human adenovirus is described in Example 4, and As illustrated in FIG. 6, this unique phagemid system is used to shut down in vivo. It is ruffling. Genomic DNA is first cloned into a phagemid vector And using the resulting plasmid (designated "Admid"), a helper M13 By using it, a single-stranded (ss) Admid phage can be produced. In vivo recombination SsAdmid fur containing allogeneic human adenovirus genome to achieve A high multiplicity of infection (MOI) is achieved in F + mutS E. coli cells using di. ssDNA is R It is a better substrate for a recombinant enzyme such as ecA. High MOI, infected ssAdmid Multiple crossovers between copies of the DNA ensure a high probability. Shuffled adeno The viral genome is produced by purification of double-stranded Admid DNA from infected cells, And introduced into a permissive human cell line to generate an adenovirus library. This Genomic shuffling strategy is a recombinant Useful for the production of denovirus variants. This means that multiple genes Screening or selection of recombinant variant phenotypes resulting from the combination of modifications enable.(3) Adeno-associated virus (AAV)   AAV is a small, simple, non-autonomous virus containing linear, single-stranded DNA. Mu zycka, Current Topics Microbiol. Immmunol. 158, 97-129 (1992) . This virus is an adenovirus or certain other virus to replicate Requires co-infection. AAV as evidenced by antibodies to the virus In addition, it is widespread in the human population but is not associated with any known disease. AAV The genomic organization is simple, containing only two genes (rep and cap). genome Contains a terminal repeat (ITR) sequence of about 145 nucleotides.   AAV-based vectors typically contain only the ITR sequence adjacent to the target transcription position. including. The length of the vector DNA increases the length of the viral genome of 4680 nucleotides. It cannot exceed the width. At present, propagation of AAV vectors is cumbersome, and vector Plasmids encoding rep and cap that provide helper functions And introduction into a host cell. Helper plasmids lack ITRs and consequently Cannot be replicated and packaged. In addition, helpers like adenovirus Viruses are often required. The potential advantages of AAV vectors are that Long-term in cells that are not always, but probably not dividing, It seems to be capable of expression. Because viral DNA is incorporated It is. Vectors are structurally simple, therefore they are adenovirus It can elicit fewer host cell responses. The current major limitation is that AAV vectors It is very difficult to grow in large quantities.     B. Non-viral gene transfer method   Non-viral nucleic acid vectors used for gene therapy include plasmids, RNA, Thisense oligonucleotides (eg, methylphosphonate or phosphorothio Eat), polyamide nucleic acids, and yeast artificial chromosomes (YAC). like this Vectors typically contain an expression cassette for expressing the protein or RNA. Including. Promoters in such expression cassettes are constitutive, cell type specific , Stage-specific, and / or regulatory (eg, such as glucocorticoids) Remont; by the MMTV promoter). Transcription is enhanced in the vector. It can be increased by inserting a sir sequence. Enhancer is a promoter Yo Cis-acting sequences between 10 and 300 base pairs that increase transcription. Enhancer Can effectively increase transcription when either 5 'or 3' to the transcription unit . These are also useful when located within an intron or within the coding sequence itself. It is. Typically, viral enhancers are used, these are SV40 enhancers. Enhancer, cytomegalovirus enhancer, polyoma enhancer, and Includes adenovirus enhancer. Enhancer sequences from mammalian systems (eg, For example, mouse immunoglobulin heavy chain enhancer) is also commonly used.   All types of gene therapy vectors also contain a selectable marker gene. Appropriate Examples of markers include dihydrofolate reductase gene (DHFR), thymidine kinase Gene (TK) or the prokaryotic gene gpt (mycophenolic acid Xanthine guanine phosphoribosyl tran selectable with (mycophenolic acid) Spherase; ne that can be selected with G418, hygromycin, or puromycin o (neomycin phosphotransferase); and selected with methotrexate DHFR (dihydrofolate reductase)) (Mulligan and Berg, Pr. oc. Natl. Acad. Sci. (U.S.A.) 78, 2072 (1981); Southern and Berg, J. et al. Mol . Appl. Genet. 1, 327 (1982)).   Prior to integration, the vector is simply a very small fraction of the DNA expressed to date. Have to go through many barriers that cannot be achieved. High levels of gene expression Limitations include: Due to nucleases present in blood and tissues Loss of vector; inefficient entry into cells; inefficient entry of DNA into the nucleus of cells And priority of DNA to other compartments; lack of DNA stability in the nucleus (limited nuclear stability) Factors can be different from factors affecting other cellular and extracellular compartments ), The efficiency of chromosomal integration; and the site of integration.   These potential efficiencies can lead to additional sequences, Included in non-viral vectors other than the expression cassette to be expressed Can be reassembled. Additional sequences confer stability both outside and inside the cell Mediates entry into cells, mediates cell nuclei And mediate integration in nuclear DNA. For example, Cell surface receptors using peptidic DNA structures or other protein binding sites Mediates the binding of the vector to serum proteins that bind to Thereby, the efficiency of DNA transfer into cells can be increased.   Other DNA sequences may shuffle specific compartments and escape or penetrate the nucleus Provides a direct or indirect preference to other compartments that are more efficient. Other DN The A site and structure, directly or indirectly, Binds to other proteins that enter it, thereby facilitating nuclear uptake of the vector . Other DNA sequences directly or indirectly affect the efficiency of integration. same An important factor for recombination integration is the degree of homology to the chromosomal sequence. And length, and the frequency of such sequences in the genome (eg, alu repeats) is there. Specific sequences that mediate homologous recombination are also important. Because embedding is Because it occurs much more readily in transcriptionally active DNA. Homologous targeting constructs Methods and materials for construction are described, for example, in Mansour (1988) Nature 336: 348; adley (1992) Bio / Technology 10: 534.   For heterologous, unorthodox and site-specific recombination, recombination is As discussed for the Toro system, recombinant cells encoded by cells (eg, , Cre / Lox and Flp / Frt systems interact with specific sites on therapeutic vectors Is done. A vector containing a LoxP site is used to control the Lo in the chromosomal DNA in the presence of Cre recombinase. Baubonis (1993) Nucleic Ac reports that it becomes incorporated at the xP site ids Res. See also 21: 2025-2029.   Non-viral vectors encoding products useful in gene therapy are available from Lipof. Injection, gene gun (biolistic), virosome, liposome , Immunoliposomes, polycations: nucleic acid conjugates, naked DNA, artificial virions, DNA Can be introduced into animals by means such as drug-enhanced uptake, ex vivo transduction . Lipofection is described, for example, in U.S. Pat.No. 5,049,386, U.S. Pat. And US Patent No. 4,897,355, and the lipofection reagent is Commercially available (for example, Transfectam^TMAnd Lipofectin^TM). Polynucleus Cationic lipids suitable for efficient receptor recognition lipofection of otide And neutral lipids include those of Felgner, WO 91/17424, WO 91/16024.   Due to the packaging virion particle size limitations, relatively small non-viral Existing viral vectors that can only insert the nucleotide sequence into the viral genome Uses naked DNA or lipofection complexes, unlike gene therapy vectors And transfer large (eg, 50-5,000 kb) exogenous polynucleotide into cells I can do it. This property of non-viral vectors is particularly advantageous. Because treatment Many genes that can be delivered by Id precursor protein (APP) gene, Huntington's disease gene), and Homologous targeting constructs or transgenes are required for efficient integration Can be done. Optionally, such large genes can be used to target cells to two or more Can be delivered as a fragment and reconstituted by intracellular homologous recombination (See WO 92/03917).   C. Gene therapy applications   Gene therapy vectors can be administered to individual patients, typically systemically (eg, In vivo by intravenous, intraperitoneal, intramuscular, subcutaneous, intracranial) or topical application Can be done. Alternatively, the vector may be a cell explanted from an individual patient (eg, Cells such as lymphocytes, bone marrow aspirates, tissue biopsies) or universal donor hematopoietic stem cells. Selection for cells delivered ex vivo, usually followed by the vector It can later be re-implanted into cells for the patient.   Important applications are especially in patients lacking both wild-type alleles of recessive genes. Treatment of congenital diseases. Vectors have the corresponding gene product in the patient The wild-type allele of the gene that allows for the synthesis of this product Introduce. Examples of recessive disorders include sickle cell anemia, beta thalassemia, phenylketonuria Disease, galactosemia, Wilson disease, hemochromatosis, severe combined immunodeficiency , Α-1-antitrypsin deficiency, albinism, alkaptonuria, lysosomal accumulation Syndrome, Ehrles-Danlo syndrome, hemophilia, agammaglobulinemia, diabetes insipidus, Resh-Nyhan syndrome, muscular dystrophy, Viscott-Aldrich disease Syndrome, Fabry disease, and Fragile X syndrome.   Another application of gene therapy increases the resistance of cells to infection by pathogenic organisms. Is to introduce a gene that causes Genes are microbes not found in the patient's genome May encode an antisense RNA to a sequence in the product. Alternatively, the gene , It may encode a protein that is inhibitory to microorganisms. Inhibited by gene therapy Examples of microorganisms that can be obtained are viral diseases (eg, hepatitis (type A, B, or C)) , Herpes virus (eg, VZV, HSV-1, HAV-6, HSV-II, CMV, and EBV) , HIV, adenovirus, influenza virus, flavivirus, ECHO virus Lus, rhinovirus, coxsackievirus, coronavirus , RS virus, mumps virus, rotavirus, measles virus, rubella Ils, parvovirus, vaccinia virus, HTLV virus, dengue virus , Papilloma virus, soft yu virus, polio virus, rabies virus, JC And arbovirus encephalitis virus) and pathogenic bacteria (eg, Mygia, Rickettsia, Mycobacterium, Staphylococcus, Pneumococcus, Meninges Bacillus, and conococci, Klebsiella, Proteus, Serachi A, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus Genus, cholera, tetanus, charcoal marks, plague, leptospirosis, and Lyme disease bacteria) Including. For example, the HIV sequences Tat and Rev (Maliam et al., Nature 338, 254 (1989)) Suitable targets for antisense RNA or RNA binding proteins.   Further applications of gene therapy will increase the selective toxicity of drugs to cancer cells in patients. Delivery of drug resistance genes to non-cancerous cells in patients with the aim of increasing . For example, polynucleotides that confer resistance to chemotherapeutic agents (eg, hALDH-1 or Alternatively, the expression cassette that drives constitutive expression of the hALDH-2 gene is a cyclophosphamide Can confer resistance to non-neoplastic cells, especially hematopoietic cells.   In another application, a negative selection gene is selected using a gene therapy vector. Delivered to the patient's cells (eg, cancer cells or pathogenic cells) where selective removal is desired. It is. Examples of negative selection genes are ricin or diphtheria toxin, and HSV Including thymidine kinase (tk). A vector having such a gene is a vector Is selectively introduced into target cells via cell surface receptors with specific affinity Can be done. The expression of the negative selection gene was determined by the presence of ganciclovir in the case of HSV tk. Kills cells carrying the gene).   In another application, gene therapy vectors provide protection to subjects at risk of infection. Or as a vaccine to treat already infected subjects. You. Such vectors encode immunogenic epitopes of pathogenic microorganisms and In patients, particularly target tissues that are initially at risk of infection (eg, , Oral mucosa and genital mucosa).III . Application of recursive sequence recombination to gene therapy   The method of the present invention relates to methods and materials (in vivo, ex vivo) used for gene therapy. Includes animals, cells, and vectors for use in vivo and in vitro systems ) Can be used to evolve or improve. This section is specific to gene therapy. Discuss the application of recursive sequence recombination for the goal of Many of these goals are The present invention relates to improvements in vectors used for gene therapy. Unless otherwise indicated, this method , Both viral and non-viral vectors.   (A) Improved titer of viral vectors   In one embodiment, the virus with improved titer is a recurrent set of the invention. It can be evolved using replacement methods. High viral titer properties are a factor in gene therapy. Of multiplying large amounts of virus for use in vitro as a drug Can be This property also indicates that the virus replicates in the host tissue, It is desired that the progeny virus infect cells surrounding the originally infected cell If useful. Virus titers can be improved by recursive sequence recombination. The first substrate for recombination is sequence divergence as a result of natural or induced modifications. May be the viral genome. Substrates can be whole or viral genomes. Part of which is known to be particularly important in giving high titers possible. The substrate can be recombined in vitro or introduced into a cell and And can be recombined in vivo. In vivo recombination is directly screened. Can be used to generate progeny viruses that can be used. However, in vitro pairs The recombination leads to a recombinant genome or a fragment thereof. The entire recombinant genome is Virus is packaged using a caging cell line or in vitro packaging system. Can be caging. Genomic fragments are usually opened first by DNA ligation. Can be assembled. These are then added to the viral genome before packaging. Inserted. Regardless of the exact pathway, at least part of the genome Virus populations with the genomes that make up these fragments.   Harvesting a virus with a recombinant genome simply involves transferring the virus for several generations in cell culture. It can be screened by subculture. This has the highest titer Viruses gain the greatest presentation among progeny viruses. If desired, Lus can be plaque purified and the titers of individual viruses compared to The best titer virus can be identified from a single recombination. Alternatively, the virus Determines the best titer virus purified by serial dilution from a single recombination I can do it.   Genomes from viruses that survive screening can be used for one more round of recombination. This can again be performed in vivo or in vitro. In vivo pairs For recombination, a virus with a genome containing the recombination segment is, for example, If so, cells can be infected at a high multiplicity. For recombination in vivo, see Will Isolated from viruses with DNA. Viruses that survived screening Genomes from each other or new substrates from sources similar to the first substrate It can be recombined. Some recombination steps are used to reduce silent mutations It is desirable to include an excess of the wild-type version of the viral genome. In addition, recombinant Can be performed on the whole genome or a fragment thereof. The selection continues as before returned.   After several rounds of recombination and selection, a viral mutant or clone that can produce the desired titer A loan can be obtained. For example, at least 10% without enrichment of the infected cell culture⁶ ,Ten⁸Or 10^TenConcentration of virus / ml evolved virus can be achieved Noh.   (B) Improved infectivity of the virus   Virus infectivity is transmitted to cells when the virus inoculum contacts excess cells Means the% of the virus you want. Obtaining high infectivity depends on the intended target cell type Is particularly important with respect to Thus, viral vectors target valuable expression products Used to deliver tissue (eg, lung cells that lack a functional endogenous CFTR gene) It is usually desirable that the highest percentage of virus infect the cell type.   The choice of substrates and means of recombination will be discussed and discussed for improved virus titers. Follow the same principle. However, screening for viruses with recombinant genomes The means are usually different. Previous selections do not necessarily select highly infectious viruses. Do not select. Because high titers are also given by the high release per cell Because it can be done. For more detailed screening for high infectivity Using a clonal isolate of the virus with a recombinant segment, separate Infect the culture. The percentage of virus-infected cells is then, for example, Determined by counting cells expressing the marker expressed by the virus Can be After several rounds of recombination and screening, 50, 75, 95, or Gives a virus with a recombinant genome that can infect 99%.(C) Improved packaging capacity of the virus   Viruses and vectors capable of incorporating increasing amounts of recombinant nucleic acid sequences (Eg, having improved packaging capabilities within the viral capsid) Can be evolved using the recursive recombination method of the invention. As mentioned above, gene therapy Commonly used viruses package only a limited genome length And thus limit the ability of the virus to accommodate large inserts. Virus Can be improved using principles similar to those discussed above. these In the method, the extended viral genome affects other viral functions Sites where increasing lengths of nucleic acids can be inserted in successive rounds of screening Should have. First, the length of the combined genome is the maximum capacity of the virus. One can start with a viral genome that has an insert that is near force. In other ways As such, the recombination starting material is a modified viral genome. Variants are usually of length Occurs outside of the insert that imparts Because this insert is This is because it is replaced in the application. One source of starting material is the evolved window. But known to display similar sequences It may be a viral genome having a carrying capacity. Recombination is as demonstrated above. Proceed in a similar fashion. The virus having the recombinant genome is then transformed as described above. Are screened for titer or infectivity. Best titer and / or feel Manipulating recombinant genomes from infectious viruses to increase genome length A further insert is introduced for the purpose. Virus can accommodate inserts of desired size This Until further achievement of recombination, screening, and increased genome length Continue the cycle. For example, in the largest adeno-associated virus vector The largest insert used is approximately 5 kb, which is 10, 15, 20, or even Or up to 50 kb or more.   (D) Improvement of virus stability   Viruses with improved stability are evolved using the recursive recombination method of the invention Can be done. The stability of the virus for use in gene therapy depends on its production and administration. Extending the life of the virus as a drug during and subsequently reaching the target Important in both the ability of the virus to resist previous cellular degradation mechanisms . The principles for the choice of starting material and the implementation of the recombination will depend on the other Same as the law. Has a recombinant genome that has evolved to achieve greater stability The virus exposes the virus to destabilizing conditions and recovers viable virus. And can be selected by For example, temperature (hot or cold), mechanical destruction (eg, , Centrifugation or sonication), exposure to chemicals or biological degradants (eg Exposure to proteases (eg, serum proteases). To destabilizing conditions The virus that survived the exposure was due to growth of the treated virus and collection of progeny. Identified. Occasionally, proliferation will progress for only one generation or a limited number of generations. What If not, then the progeny virus has a genome with a conferring genome. Because the virus is biased to have a genome that favors high titers in addition to ills. You.   (E)Improved expression or regulation of expression of vector coding sequences   Improvement of the expression of the target gene sequence can be achieved by implementing the recursive sequence recombination method of the present invention. And can be achieved. Viral vectors usually used in gene therapy Alternatively, non-viral vectors contain products expressed in the intended target cell. To load. The product can be a protein or RNA, for example, an antisense RNA or Or RNA that specifically binds to the target protein (ie, an aptamer) You. Usually, a coding sequence is operably linked to a further sequence (eg, a control sequence). To ensure its expression. For example, some or all of the following sequences: : Enhancers, promoters, signal peptide sequences, introns and / or Or a polyadenylation sequence. The desired purpose is achieved with a conventional vector To increase the expression level of a functional expression product as compared to the expression level . Expression can be achieved by various means (increase in production rate of expression product, decrease in degradation rate of expression product). Or improved expression product's ability to perform its intended function) Can be improved.   Improved expression of the selectable marker is achieved by performing recursive sequence recombination. obtain. For selection purposes, gene products expressed from vectors are sometimes Markers that are more easily detected than products with actual therapeutic purpose (eg, Fluorescent protein (see Crameri (1996) Nature Biotech, 14: 315-319). )) Or a cell surface protein. But some with therapeutic purposes The gene (eg, a drug resistance gene) itself provides a selectable marker and No need for or replacement markers. Alternatively, the gene product is It may be a fusion protein comprising any combination of Kerr and a selectable marker.   The material for recombination can operate on the coding sequence and / or the coding sequence It may be a full-length vector containing a control sequence linked to or a fragment thereof. This Of any of the control and / or coding sequences present in the vector It may include variants. If recombination is achieved at the fragment level, The fragment should be reinserted into the vector before screening. Recombination When the vector proceeds in vitro, the vector containing the recombinant segment is usually screened. It is introduced into cells before leaning. Cells containing the recombinant segment are Screened by detecting the expression of the gene encoded by Kerr obtain. Internal reference marker gene detects mutations in copy number or insertion site And can be included in the vector to correct. For example, if this marker is green If it is a fluorescent protein, cells with the highest expression levels^TMBy Can be identified. If the marker is a cell surface protein, the cell Stained with a reagent (eg, an antibody) that has affinity for^TMBy Analyzed. Recombinant segments from cells with the highest expression are Used as some or all substances in leaning.   Development of cytomegalovirus transcription control elements   Major immediate early (IE) region transcription control elements of cytomegalovirus (CMV) (Promoter sequence and enhancer sequence (promoter / enhancer region ) Regulates transcription in vectors used for gene therapy Widely used for. Because this is very Because it is active. Optimized CMV for increased transgene levels Transcriptional control elements are produced by the recursive recombination method of the invention, Improve the efficacy of treatment. CMV promoter and enhancer are used in human cells and Improved CMV promoter / enhancer as active in animal cells Use elements to control foreign genes in both animal models and clinical applications. Is expressed.   A library of chimeric transcription control elements was derived from five related strains of CMV. Produced by DNA shuffling of wild-type sequences. IE region promoter sequence, The enhancer sequence and the first intron sequence were obtained by PCR from a CMV strain. Biol. Med. 92: 418; Human V-97 strain AD169 (Rowe (1956) Proc. Soc. Exp. Biol. Med. 92: 418) 7 Towne (Plotkin (1975) Infect. Immunol. 12: 521-527); Rhesus monkey VR-677 strain 68-1 (Asher (1969) Bacteriol. Proc. 269: 91); Velvet VR-706 strain CSG (Black (1963) Pro c.Soc.Exp.Biol.Med.112: 601) and squirrel monkey VR-1398 strain SqSHV (Rangan (1980) Lab. Animal Sci. 30: 532). The promoter / enhancer sequence of the human CMV strain It is 95% homologous and 70% homologous to the sequence of the isolate. This is live Enables the use of family shuffling to generate a great variety of rallies I do. After shuffling, the library was cloned into a plasmid backbone and This is used to direct transcription of the marker gene in mammalian cells. Internal markers under the control of the native promoter can be included in the plasmid vector. You. Expression markers such as green fluorescent protein (GFP) and CD86 (B7.2 Freeman (1993) J. Exp.Med. 178: 2185, Chen (1994) J. Immunol. 15 2: 4929) can also be used. In addition, SV40T antigen-transformed cells Transfection can be used to amplify a vector containing the SV40 origin of replication. Transfected cells are those that express high levels of the marker gene. To account for differences in vector copy number per cell Screened by FACS classification normalized to internal markers. As desired If so, recover the optimal vector containing the recurrent recombination promoter sequence, and For further cycles of shuffling and selection.   (F) Improved activity of drug-resistant enzymes and expression of drug-resistant sequences   The recursive recombination method of the invention also improves the expression of drug resistance sequences / proteins To provide the means for: Many treatment regimens rely on side effects on specific cell types in the body. Administration of a drug having a specific purpose. For example, chemotherapy can Notorious for killing cells. Llcht (1995) Cytokines & Molecular Therapy 1: 1 See 1-20. Myelosuppression, or bone marrow toxicity, is associated with many chemotherapeutic agents. To limit the capacity. This is not only a dangerous side effect, but also the effectiveness of chemotherapy Also restrict. In fact, chemotherapy can be performed directly or by leukemia due to a loss of blood cell function. Can be indirectly fatal by producing secondary cancers such as Gene therapy Can protect hematopoietic cells by delivering drug-resistant proteins via Noh. This principle has been demonstrated by numerous studies. Where the mouse bone marrow Cells are treated with a protective alkyltransferase for chemotherapy alkylating agents. Protected by overexpression. Other drug resistance proteins protect the normal tissue And a target for improved expression using the methods of the invention. Can be. These include, for example, glutathione-S-transferase, dihydrogen Lofolate reductase, and superoxide dismutase.   Alkylating agents are particularly toxic to the hematopoietic system, With myelosuppression. Hematopoietic cells are very sensitive to alkylating agents. Thus, iatrogenic leukemia is a common symptom. Alkylation treatment can also cause severe lung poisoning Can occur. This limited sensitivity is associated with the DNA repair protein O in hematopoietic cells.⁶- Methylguanine-DNA methyltransferase (which is also⁶-Alkyl gua Nin-DNA alkyltransferase, MGMT, or alkyltransferer Ze; EC.2.1.1.63). Alkylating agents, especially nitrosoureas, Used alone or in combination with other drugs, many types of cancer, for example, Hodgkin's and non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, brain tumor Treat ulcers, gastrointestinal pills, and lung cancer. Taken together, these are all It constitutes more than one-third of cancers diagnosed. Therefore, the alkyl agent base Of Improving the effectiveness and reducing toxicity of chemotherapy regimens has been It has a profound effect on treatment.   Via gene therapy of bone marrow stem cells or lung cells for drug resistance genes such as MGMT Is one way to overcome the therapeutic limitations imposed by the toxicity of chemotherapeutic agents. Is the way. One strategy is to explant the patient's hematopoietic progenitor cells or lung cells ex vivo. Transduce and grow bone marrow using these cells before or after chemotherapy It is. See generally Dunbar (1996) Annu. Rev. Med. 47, 11-20. Bone marrow A tissue that is relatively easy to extract, manipulate, and reintroduce into the body. Lung Vesicles can also be extracted, manipulated, and then reintroduced by an inhalation delivery system. Alternatively, the gene may be delivered by a spray delivery system (eg, Eastman (1997) Hum. Gene Ther. 8 : 765-773 (1996) Hum. Gene Ther. 7: 1701-1717 Aerosol delivery of transgenes using DNA; or adenovirus vector For delivery of sols, see MacDonald (1997) Hum. Gene Ther. 8: 411-422) It can be introduced directly into alveolar cells more in vivo.   MGMT has been found in all tested organisms and is a chemotherapeutic alkylating agent. Prevent mutagenic, cytotoxic, and carcinogenic effects of MGMT, O of guanine⁶ The alkyl group bound by such chemicals is removed from the position. These The alkyl group is irreversibly transferred to cysteine in the active site of the MGMT protein. And inactivate the alkyltransferase. Therefore, this enzyme is a suicide enzyme And can only work stoichiometrically. This is important for MGMT improvement Barrier. Each protein module acts only once in a suicide manner, The protection afforded depends not only on the activity (quality) of MGMT, but also on the number of MGMT molecules. It is determined. Little or no alkyl transferers, such as bone marrow cells Cells that do not express the enzyme can be used with laboratory alkylating agents (eg, N-methyl-N'-nitro -N-nitrosoguanidine (MNNG)) (Day (1980) Nature 288: 724-727) and clinical Very sensitive to nitrosourea (Erickson (1980) Nature 288: 727-729) used in High in nature. Therefore, myelosuppression is an important part of alkylation-based chemotherapy regimens. Serious problem (DeVita (1993) Cancer: Principles and Practice of Oncology) Has a wild-type human, mouse, or bacterial alkyltransferase gene It has been overcome in experiments transducing human and mouse hematopoietic cells. Leto Overexpression of genes carried on rovirus vectors can Was protected from killing by nitrosoureas (Allay (1995) Blood 85: 3342-3351: Mori tz (1995) Cancer Res. 55: 2608-2614). In addition, these cells are found in the bone marrow of mice. When implanted, protection has been demonstrated to be long lasting in vivo (Maze (199 6) Proc.Natl.Acad.Sci.USA93: 206-210). A similar effect occurs on the liver and the bone rather than the bone marrow. (Dumenco (1993) Science 259: 219-222) And Nakatsuru (1993) Proc. Natl. Acad. Sci. USA 90: 6468-6472).   This protective effect of MGMT can be improved in some ways by recursive sequence recombination. First, the higher the specific activity (ie, the faster the cytotoxic alkylation-induced damage New variants with good repair) can be selected. Therefore, for a given expression level In addition, bone marrow cells are better protected. MGMT using traditional cassette mutagenesis Have been reported (Christians (1996) Proc. Natl. Acad. S. ci.USA 93: 6124-6128). Second, the new variant is⁶-Like benzylguanine For wild-type alkyltransferase inhibition. This Inhibitors, such as, may sometimes endogenous alkyltrains present in cancer cells. Used to inhibit phosphatase (Pegg (1995) Progress in Nucleic Acid Res. and Molec. Biol. 51: 167-223). Using inhibitor resistant MGMT Combines an alkylating agent with an alkyltransferase inhibitor Bone marrow is transfected in the treatment protocol. Third, the code sequence And / or a novel variant of an operably linked control sequence may be used to improve expression of MGMT. You can choose for good. Fourth, it binds to but removes alkyl adducts from DNA. A variant of MGMT can be selected that is more effective against cells than the alkyl adduct alone. This results in a more toxic DNA-protein crosslink. Vectors expressing mutant variants Can be targeted to cancer cells prior to treatment with an alkylating substrate. Fifth, MGMT variants may protect mammalian cells against clinically relevant nitrosoureas Can be selected for For this purpose, the selection should be a mammalian rather than a bacterial cell. It should preferably be performed in animal cells. Because nitroso urine Because the protective effect of MGMT on the element is stronger in mammalian cells . Small If not, the last round of selection is usually performed on stem cells. Because of drug resistance Because some component factors that contribute to the endpoint may be cell type dependent.   Since the expression level is important for the protective effect, other than the sequence encoding MGMT Manipulation of vector sequences provides an important source of improvement. The vector is used for the desired purpose (eg, For example, the ability to protect cells from alkylating agents). The purpose is Various components and combinations that are too complex to determine (enhance transduction, Including improved gene stability in addition to improved MGMT activity) Is achieved by   Low transfection efficiency as in gene therapy Is not a major limitation in Because the alkylation process is transfected Because it effectively acts as a positive selection for the selected cells. Contrast In addition, low transfection efficiency is a problem in in vivo gene replacement therapy. possible. Because there is no general positive selection, tumor killing gene therapy This is because only the negative selection by exists. About in vivo gene replacement therapy Improvements in the means of positive selection have e.g. To regrow into bone marrow.   The drug resistance gene as a starting material for the improvement using the method of the present invention is a multi-drug It is the resistance gene MDR-1. MDR-1 is a plasma called “P-glycoprotein (Pgp)” Is a membrane glycoprotein that acts as an ATP-dependent drug efflux pump and Confer chemical resistance to a wide variety of drugs (Chin (1993) Adv. Cancer Res. 6 0: 157). Cells that do not express MDR-1 contain vincristine, ethoxide, and It is brilliantly sensitive to drugs like luchicine. Expressed by tumor cells This similar chemoresistance property, which can ruin the chemotherapy effort in some cases, is positive When used as a selective marker, it can be advantageous. Metz (1996) Virology 2 17: 230-241 is 20 times higher when selecting for MDR1 expression compared to neo selection. Reported the stringency. P-glycoprotein has at least two different genetics Child disease, Fabry disease (Suglmoto (1995) Human Gene Therapy 6: 905-915) In vitro correction of cells from granulomatous polyposis (Sokolic (1996) Blood 87: 42-50) Positive selection has been demonstrated for transformed cells. I But why do we think nature has optimized MDR1 for activity against artificial drugs? not exist. Improvement of MDR-1 by recursive recombination to etoposide and colchicine By improving the protection of cells from drugs such as, higher levels of this Allows the use of such selective agents, increases selective stringency, and Better differentiate between transgenic and non-transformed cells.   MDR-1 can be used for positive selection in mammalian cells following DNA shuffling. And is improved / modified. A pool of MDR-1 random mutations can be transferred to an appropriate vector (eg, E.g., retroviral vectors, adenoviral vectors) Transform into resistant cells. Colchicine and / or etoposide and / or Alternatively, selection with vincristine identifies active MDR-1 variants. MDR-1 gene Rescue from living cells, and additional rounds of recombination with increasing doses of drug And offer for selection.   Some mammalian cells already express high levels of P-glycoprotein Therefore, it was determined whether the improved MDR-1 transgene was expressed in these cells. It can be difficult to determine (ie, the background is high). This place In some cases, endogenous P-glycoprotein is a well-characterized inhibitor (eg, Rapamil) and inactivated by the inhibitor but still cytotoxic MDR-1 encoding a mutant P-glycoprotein that is highly active against substances Transform with the transgene. Such variants may be inhibitors and cytotoxic Select pool of MDR-1 mutants in the presence of both sex drugs (eg, colchicine) Is selected by For example, using the methods of the invention, cytotoxic chemicals Superactive against N-methyl-N-nitro-N-nitrosoguanine (MNNG) Completely resistant to the benzyltransferase inhibitor benzylguanidine Create an alkyltransferase variant.   Therefore, MDR-1 optimized as a positive selection marker was Insert into the connector. Vectors can also contain DNA shuffling, itself or MDR-1 variants. In combination with allogeneic induction (shuffled unit MDR-1 and vector) Can be optimized. By shuffling the entire construct, many parameters It is possible to test at one time. Bicistronic sequence (from the same promoter) Two transcribed as one mRNA but transcribed from separate ribosome binding sites Gene can be used (Sugimoto (1995) Human Gene Ther. Supra). Up to the entire array Or shuffling the entire construct to translate the second downstream gene The secondary structure of the improved cistronic mRNA can be optimized. For example, MDR-1 and Bicistronic retroviral vector encoding a gene that complements a genetic defect Can be constructed and optimized using the methods of the present invention. The whole vector is , Can be mutagenized by DNA shuffling and rearrangement. In addition, the vector Can be packaged as a virus by a packaging cell line, It can be transfected and selected with colchicine. Selection is for surviving cells Achieved by analyzing for complementation of a genetic defect.   MDR-1 protects against drugs it already recognizes (eg, etoposide). Not only provide improvements, but also for drugs that are not recognized by wild-type MDR-1 It can be evolved / modified to provide protection. For example, recursive recombination of the MDR-1 gene Can be modified (evolved) to excrete the alkylating agent out of the cell, and therefore MGMT (hereinafter (Described above). For example, both MGMT and MDR-1 genes To stem cells prior to combination chemotherapy (one of the drugs is an alkylating agent) Can be transformed. Studies that have transformed stem cells with the wild-type MDR-1 gene have MGMT for the killing agent gave similar results to the above (Sorrentino (1992) Science 257: 99).   Another suitable gene for evolution / modification using the methods of the invention is glutathione S -Transferase, which detoxifies alkylated drugs in the cytoplasm, Complements GMT. It acts on the drug after it enters the nucleus and damages the DNA I do. Glutathione S-tran obtained from conventional cassette mutagenesis in bacteria Some improvements in spherases have already been reported (Gulick (1995 ) Prod. Natl. Acad. Sci. USA 92: 8140-8144). Further by recursive sequence recombination Evolutions provide further improvements. The improved gene is then Or transfected into stem cells or lung cells in combination with MGMT obtain.   Other drug resistance genes used in suppressing side effects in other tissues About being a candidate for evolution. For example, bleomycin is a major poison An anti-malignant pulmonary drug whose sex is to lung cells. Bleomycin protein Hydrolytic enzymes can protect cells from bleomycin, and human genes have It was cloned (Bromme (1996) Biochemistry 35: 6706-714). Genes are left Improved by gene shuffle and for protection of lung cells in cancer patients Can be used.   In other embodiments, candidate genes for improvement include DNA ligase and Topoisomerase for protection from ionizing radiation (Boothman (1994) Cancer Res. 54 : 4618-4626), T4 to protect against UV radiation and skin cancer A gene encoding a nucleotide cleavage repair enzyme such as endonuclease V, And base cleavage repair pathways (which cause many types of cancer and accelerate aging) Is thought to be important to prevent the effects of oxidative DNA lesions) Encoding alkaline phosphatase endonuclease and glycosylase Gene.   Evolution / modification of drug resistance genes and related regulatory sequences using the methods of the invention Are under the general approach described above for improving gene expression. But, Sometimes improving gene expression in the evolution of drug resistance genes Not only increase the degree of resistance conferred by the gene product with a particular drug. Is desired. In this situation, the genetic situation in which the resistance gene should be expressed In order to be able to evolve in It is preferred to include the regulatory sequences described. The initial intersubstrate diversity depends on the induced mutation. Provide natural drug resistance genes from various sources, and improved properties. May be the result of a known mutation.   For example, five different mammalian species of MGMT (human, rat, mouse, hamster , And rabbits) have very broad homology (mutations) as shown in FIG. Despite the body being present) has been reported. The following is the top stage Amino acid sequence, an alignment that shows other amino acid sequences that occur naturally under the human sequence It is.   Natural modifications are specific to human and non-human forms, as discussed in Example 3. To create a recombinant form of MGMT containing a natural segment, as discussed in Section II. It can be incorporated in any format. For example, the oligonucleotide may be a natural variant Can be designed to code all the different combinations of The gonucleotide is mixed with the fragmented wild-type human gene. Surprise In short, a small number of oligonucleotides (21) have more than two amino acids naturally If degenerate at existing locations, it can be used (see FIG. 3). Shown in FIG. Oligonucleotides are based on a 20 base sequence that perfectly matches the human MGMT sequence. Includes up to 21 bases that are not homologous to the human sequence flanked on either side. "Oligo Spa Another use of "iking" has been demonstrated to improve wild-type proteins ( Christians (1996) Prod. Natl. Acad. Sci. USA93, 6124-6128) V139F mutation or Or O⁶-For known desired mutations such as those that confer benzylguanine resistance To bias the shuffled gene pool.   An alternative to “oligo spiking” is to obtain all individual cDNAs and mix them It is a combination. This selection avoids pooling when used for human gene therapy. It has some tendency to alleviate human qualities leading to epidemiological problems, The problem is that you reverse the mutant with the wild-type human gene to eliminate It can be overcome by inserting.   The recombinant drug resistance gene and the vector encoding it have been improved. Can be easily screened for expression. Vector containing recombinant segment The cells containing the cells are exposed to the drug and the recovered viable cells. These cells Are rich in recombinant segments that confer improved resistance to drugs. School Leaning is achieved by increasing drug concentration or exposure time It can be made more stringent in a typical round.   (G) Transduction vectors for integration and stable expression in mammalian stem cells Evolution   Novel and novel methods for expressing itself in infected, integrated, and hematopoietic stem cells And / or a vector having improved ability can be prepared using the recursive recombination method of the present invention. And evolve. Key goal of gene therapy is to effectively construct DNA in human stem cells Is to evolve a practical way to incorporate. Retrovirus on stem cells Practical methods for effectively incorporating DNA are genetically modified with the desired properties. Enables repopulation of patients who have autologous bone marrow transplantation. For example, The vesicles are engineered to express a trans-dominant factor that interferes with viral replication. stem Complement cells with wild-type or defined genetic deficiencies (eg, Gaucher disease) Are engineered to express the engineered transgene. MDR gene or al The califortransferase gene is stemmed to render it resistant to chemotherapeutic drugs. Insert into cells. T cell receptors specific for a cancer or pathogen epitope of interest Is inserted for expression during stem cell maturation.   However, stem cells are difficult to purify when grown in vitro, and Rapidly eliminates the pluripotent phenotype. Retroviruses are generally non-dividing cells. Very inefficient in integration into cells (especially stem cells). Therefore, one factor Or a set of factors (such as infection with a retroviral gene before integration and its In making the stem cells temporarily or permanently susceptible to retroviral integration, Uses recursive recombination used to evolve (while remaining pluripotent at the same time) I do.   In one embodiment, expressing a candidate factor for temporarily disrupting stem cells Large libraries (> 10) that promote retroviral integration⁶)of Create a retrovirus. Such factors include the HIV matrix, HIV vpr, Random fragments of HIV, and other lentiviruses (effective against non-dividing cells) The only class of retrovirus that can be introduced into the cell); cDNA from stem cells; stromal cell culture Nutrient-derived cDNA, which makes factors that affect the state of stem cell differentiation, and Production or evolution of the morphology exerts the desired effect); or any other site Examples include, but are not limited to, cain or growth factors. Such a lie The library can be used to generate in vitro and in vivo regressions of the invention, generally as described above. Target sequences and proteins in non-dividing stem cells Create retroviruses that can be infected, integrated, and expressed at a high rate.   According to another embodiment, the retrovirus is modified by the method described above. Retransfect transgenic SCID or SCID / NOD mice with human stem cells . Successful by member of first retroviral recombination segment library Retroviral progeny from the successfully transduced stem cells are recovered. Selection marker -(Eg, green fluorescent protein (GFP), drug marker, or cell (FACS) marker), and the retrovirus Recovery of stem cells transduced with the DNA construct may be facilitated. Evolved / modified Sequence encoding the factor to be integrated or the entire integrated retroviral genome. Can be recovered. Perform additional rounds of recursive sequence recombination at the desired efficiency / stem cell transduction Can be repeated until the efficiency is achieved.   A mouse SCID / NOD immunodeficient system that can repopulate primitive human hematopoietic stem cells can be used (Dick (1996) CSH Gene Therapy, summary # 11). Retroviruses use these Stem cells can be infected with very low but detectable efficiency. Retro retro Progenitor cells from peripheral blood cells in this SCID / NOD repopulation model I can do it. Therefore, this regrowth and similar regrowth systems are transformed into primitive pluripotent cells. Form the Basis for Selecting Retroviruses with Improved Efficiency of DNA Integration I do. As mentioned above, the inclusion of GFP in the vector will result in retroviral Enables FACS purification of cells expressing Rus-encoded protein. Regrowth first If very inefficient, selectable sources for selective culture of transduced cells The gene (eg, Neo or TK) is also expressed.   In another embodiment, the infected stem cells are removed and additional recurrent recombination is performed. Lethal with recombinant sequences rather than isolating retroviral sequences for one time The irradiated retrovirus-producing helper strain is injected into the SCID / NOD bone marrow. This Recursive recombination takes place in vivo using the technique of Stem cells, a special environment of the bone marrow. (An environment that can prove incapable of mimicking in vitro) Continue.   In a further embodiment, recursive recombination is used to integrate into non-dividing cells. Develop a virus-based means that cannot be properly eliminated. This method is HI HIV vectors, which are required for V to integrate into non-dividing cells, can be Incorporate in For example, integrase (a viral genome in the host cell chromosome) Enzyme that mediates cell integration, imports the HIV-1 pre-integration complex into the nucleus It may be sufficient to couple with the mechanism. Viral matrix and Vpr protein Quality also plays an important role in the ability of HIV to integrate into non-dividing cells. Galla y (1997) Proc. Natl. Acad. Sci. USA 94: 9825-9830. DNA shuffle A repetitive cycle of recursive recombination, such as full, can be performed on the desired vector or target Is performed until it is given to the sequence.   In another embodiment, long-term bone marrow cultures are stimulated prior to recurrent recombination. Cycle in vitro. This is true for both mouse SCID / Beige regrowth assays. (Agatsuma (1997) Antiviral Res. 34: 121-130). Stem cell repopulation in late-stage human bone marrow lung patients Produces irrus transduction. Cycle stem cells are more sensitive to transduction . Thus, stem cells can be stimulated and more susceptible to retroviral transduction. And still maintain pluripotency.(H) Improved tissue specificity of vectors   Vectors with new and / or improved tissue specificity (tissue tropism) It can be evolved using the recursive recombination method of the invention. For most gene therapy applications Where gene therapy vectors are delivered to specific tissue types with high specificity. Hope. Specific cellular targets can be used in these vectors for in vivo gene therapy. This is an important issue that affects the safety and practicality of a computer. Therefore, specific genetic Limit the specificity of the offspring therapeutic vector (eg, adenovirus) and / or There is a need to redirect.   One example showing the need to deliver gene therapy vectors to specific tissue types is Delivering the type CFTR gene to a cystic fibrosis patient. The CFTR gene is mainly Should be delivered to the lung tissue. In a second example, the gene therapy vector is When coding for a chemotherapeutic drug, the drug is directed at neoplastic cells rather than normal cells. It is desired to be delivered.   Strategies for substrate selection and recombination format selection are generally This is similar to the strategy described above. Substrates for recombination span the entire viral genome Viral proteins that are likely or that are thought to interact with cellular receptors It may be a fragment encoding the protein. Such fragments are recombined If recombined, the recombinant product is reinserted into the viral genome and the genome is encapsulated. Should form a virus before screening.   For example, the development of vesicular stomatitis virus (VSV) to infect new target cells Regression should focus on G protein sequences . This is because G proteins are expressed on the outer surface of the capsid (Schne ll (1996) Proc. Natl. Acad. Sci. USA 93: 11359-11365). In addition, bullous mouth Generating a virus encoding the endogenous virus G protein (VSV-G) is a technique It is technically difficult. Because it is highly toxic to host cells, This is because breeding is impossible (Yoshida (1997) Biochem. Biophys. Res. Commun. . 232: 379-382). Therefore, the method of the present invention is used to produce a modified VSV G protein. Used to generate new target cells for recombinant VSV.   There is also a need to generate tissue-specific adenovirus. Adenow Tissue-specific expression of systemically administered vectors due to non-selective illness tropism Is a tissue-specific promoter / enhancer (this is small enough to Can be achieved only by the use of Alternatively, tissue specific Expression can be used to eliminate the natural promiscuous tropism of adenovirus, and the method of the invention. It is generated by construction of a new tissue-specific domain to be used. For recursive sequence recombination Tissue-specific adenovirus production is a key to the use of vectors in gene therapy. To overcome this non-selective tropism limitation of the native adenovirus.   Adenovirus protrudes from each of the 12 vertices of its icosahedral capsid It binds to eukaryotic cells using "fiber proteins". Serological and mutagenic Research has shown that a fiber homotrimer consisting of "staff" and "knob" domains Reveals interaction with cellular receptors. The knob structure is Xia (1994 ) Reported by Structure 2: 1259-1270. R. D. Gerard, hetero bird The structure of the meric knob was modified for mutations that decrease receptor binding. Used to scan by SDM (Private Communications, 1996 CSH Gene Therapy meeting) Discussion). These studies have shown that natural receptors (known to be expressed on many tissue types). Have the ability to eliminate or significantly reduce the ability to transmit Allows construction of mutants. This is an adenovirus that binds to cellular receptors Starting point to develop a new tissue specificity for fiber , Using the recursive sequence recombination method of the present invention). V. Legrand (CSH poster 1 84) and Dan von Seggery (CSH Poster # 223) are small, easily manipulated S V40-based vector reports a system for expression of mutants off fiber proteins . These constructs are responsible for plaque formation by adenoviruses lacking the fiber gene Instruct. Lcgrand produces 11 amino acid gastrin releasing peptide (GRP) This system was used to fuse to the C-terminus of the gene. Express this fusion protein LacZ + adenovirus mutant has a soluble knob protein (CSH poster 184) Although only 60% can be inhibited, the virus that expressed the wild-type protein Cells that express the GPR receptor can be infected in a manner that is about 90% inhibitory. This It states that the interaction of GRP with its receptor supports infection of host cells. Given as evidence.   In one exemplary embodiment, the adenovirus is expressed using a method of the invention. Binds to the mutant fiber protein or its natural receptor to improve the system Create a domain that replaces a knob that lacks the ability to do so. Evolved by recursive recombination Of a novel fiber sequence has a novel adenovirus fiber, or a new specificity To produce a knob binding ligand. Alternatively, the library of mutated sequences is It can be inserted at the C-terminus of the knob in a manner similar to the GRP construct. Potential ligand Library randomized throughout the "staff" and / or "knob" domains Can be inserted. The entire knob was randomly mutagenized and infected with the desired target. You can choose to Other exposed viral proteins (eg, penton or Exon protein) can be mutated in a library of insertional variants. Short protein Library coding for the adenovirus hexon protein And can be expressed on the surface of adenovirus virions as part of a hexon ( Crompton (1994) J.C. Gen. Virol. 75: 133-139). Then these recursive progress Infect target cells with these mutated viruses, including mutated viral proteins Use to In viral proteins that affect adenovirus tropism Diversity and modification by plaque formation or by cell sorting (FACS) May be based on the transient expression of a reporter gene (eg, GFP) Select.   Integrin (α-v-integer) on the target cell surface of fiber penton protein Interactions with cell-virus stabilizing interactions (soluble fiber knob proteins) It is known that proteins cannot completely inhibit adenovirus interaction )I will provide a. In the absence of fiber penton-cell integrin interactions, Luss infection is low. Mechanisms determining cell specificity for adenovirus As a result of this complexity in cosmism, its cognate receptors (eg, For example, interaction with the target alpha-v-integrin penton fiber domain) To evolve multiple genes or domains in the adenovirus used The method of the invention is used for this. Therefore, the selected viral gene or virus Whole recursive sequence recombination is used to rapidly evolve tissue-specific adenovirus A particularly useful tool.   In another exemplary embodiment, the highly evolved M13 technology is Used to develop peptide ligands for the receptor of interest. Standard Ligand display library technology can be used to bind peptide ligands to purified receptors Used for screening. Alternatively, the library can be It can be screened for panning. The affinity of these ligands To evolve quickly. The pool of developed ligands is then (Eg, C-terminal fusion of fiber proteins). This is a Incorporates the power of M13 selectivity against the denovirus system, which can be created by M13 alone Not possible to make a library of this size.   The virus containing the recombinant segment is removed from the first cell desired to be infected by the virus. By contacting the cell population and a second cell population for which infection is not desired. Achieve leaning. The viral genome recovered from the first cell population is The recombinant segments that confer specificity on the cell type are abundant. First and second of cells The population can be in different tissues of the organism. For example, turning whole organisms into viruses More infected, and a subset of blood cells, which is the cell type for which infection is desired ) Can be recovered. Alternatively, a natural or derived moth Infect whole organisms, including humans, affected by the virus and remove cancer cells. From which the recombinant segment can be recovered. In a further modification, the first and second cells The cell population is co-cultured with the virus in a mixed cell culture. Two cell types Markers (green if they are not easily distinguished by microscopy) Fluorescent proteins or cell surface receptors on certain cell types) I can separate. In the first round of screening, the host cells present are usually (Eg, a ratio of 10% desired host cells to 90% present host cells). . The desired target cell population can be expanded in successive rounds.   The recombinant segment recovered from the population of cells desired to be infected is Used as a substrate in Perform subsequent screenings on the same principle .   In a modification of the above approach, a eukaryotic or bacterial virus is Ligand as a fusion protein with coat protein on the outer surface of virus Expression alters it to have specificity for a given cell type. Ligands are applied to receptors known to be present on the cell type of interest. Choose to have affinity. For example, the EGF family of proteins , Some polypeptides (eg, epidermal growth factor (EGF), Growth factor α (TGFα), amphiregulin (AR), And heregulin (HRG-β1)), which interact with membrane receptors. Regulates growth in breast cancer cells through interactions. Han (1995) Proc. Natl. A cad. Sci. USA 92: 9747-9751, Moloney murine leukemia virus fused to gp70 That can be modified to express modified human heregulin, and recombinant virus Infects Specific Human Breast Cancer Cells That Express the Human Epidermal Growth Factor Receptor Reported that.   This principle allows viruses and receptors that express ligand fusion proteins It can be extended to other pairs of expressing target cells. For example, filamentous phage is substantially Have specific binding affinity for any selected cellular receptor Can be engineered to display an antibody fragment (eg, Fab or Fv). The binding specificity of the ligand for the receptor is determined by the viral genes encoding the ligand. By recursive recombination of the nome segment, and by And by screening with a second population.   Viral vectors are used to obtain new or altered tissue specificity. Most sensitive to evolutionary / recurrent recombination, but the same principles apply to non-viral vectors Can be done. Such vectors are advantageous for uptake by specific target cells It can be manipulated to contain a specific uptake sequence that is believed to be. Or non- Variants of viral vectors recombine without prior knowledge of sequences that can mediate uptake I can do it. For example, the starting substrate can be a random sequence. The recombinant product is First and second populations of cells, and those intended for the use of the vector. Contacted under similar conditions. For example, vectors packaged in liposomes If used, the recombinant segments packaged as liposomes Screening is performed with a vector containing Again, the recombinant segment The containing vector is recovered from the population of target cells, and these segments are , Used in the next round of recombination.(I) Improved DNA uptake mediated by evolved DNA binding proteins   The uptake efficiency and specificity of vector nucleic acid uptake by a given cell type Evolved / regressively recombinant and modified proteins that bind to acids Can be improved by coating the reactor. The vector is a modified tamper Can be contacted with the protein in vitro or in vivo. In the latter situation, In the cells containing the vector, the coding sequence is optionally contained in the vector. Is expressed from Normally evolved nucleic acid binding proteins have nucleic acid binding activity Does not necessarily have any known ability to enhance or alter nucleic acid DNA uptake. No.   In this embodiment, the DNA binding protein modified by the method of the invention Include transcriptional regulators, enzymes involved in DNA replication and recombination (eg, recA), And proteins that act on structure and function in DNA (eg, histones, pro Tamine). Other DNA binding proteins include the phage 434 repressor, phage lambda cI and cro repressor, coli CAP protein, myc, leucine Zippered proteins and DNA binding basic proteins such as fos and jun Main; proteins having a “POU” domain (eg, Drosophila pairing protein) Protein having a domain whose structure depends on chelation of metal ions Proteins (eg, CyS found in TFIIIA)_Two-HiS_TwoZinc finger looks at yeast Gal4 Zn as issued_Two(Cys)₆Cluster, retrovirus nucleocapsidtan Cys found in protein_ThreeHis box and nuclear hormone receptor protein Zn found in quality_Two(CyS)₈Cluster); phage P22Arc and Mnt repressor (Knight (1989) J. Biol. Chem. 264: 3639-3642; Bowie (1989) J. Biol. Chem. 264: 7596-7602). RNA-binding proteins are available from Burd (1994) Science  265: 615-651, and includes HIV Tat and Rev.   As in other embodiments of the invention, the improved DNA binding protein Or the evolution towards acquisition of altered uptake efficiency, Produced by a recursive cycle of leaning. The starting substrate is natural Or a derived variant of one of the nucleic acid binding proteins as described above. It can be a nucleic acid segment that encodes a protein. The nucleic acid segment is a vector Or in an isolated form for the recombination step. Recombinant Can be advanced by any of the formats described in Section II.   For screening purposes, the recombinant nucleic acid segment is Inserted into the vector (if not present in such a vector) Should. Vectors are selected for expression in the cell type in which uptake is desired. Code the selection marker. Evolved DNA binding proteins are specific binding sites (Ie, the lacI binding protein recognizes lacO) A binding site can be included in the vector. If necessary, the vector may be The site may be included in tandem.   Vectors containing different recombination segments can be introduced into host cells (usually E. coli). Transformed, the recombinant protein is expressed, and encodes its genetic material Ligated into vector. Most cells take up only a single vector and Such transformation results in a population of cells, most of which Containing seeds. After an appropriate period to allow expression and ligation, the DN of the vector Lyse cells under mild conditions that do not disrupt binding to A-binding protein. For example , PH 7.5 with 35 mM HEPES KOH, 0.1 mM EDTA, 100 mM Na glutamate, 5% glycerol Rolls, 0.3 mg / ml BSA, 1 mM DTT, and 0.1 mM PMSF) and lysozyme (10 A lysis buffer of 0.3 ml at mg / ml) is suitable (Schatz et al., US Pat. No. 5,338,665). No.). The complex of vector and nucleic acid binding protein is then recovered. Improved or modified uptake is desired under conditions favorable to uptake (Eg, for eukaryotic cells, the recipient Cells can be treated with calcium phosphate or electroporated Can be provided). Suitable recipient cells are discussed elsewhere in this application. Human cell types, which are common targets in gene therapy.   After incubation, it is present in the vector containing the recombinant segment Plate the cells, selecting for expression of the selectable marker. Expressing the marker Collect the remaining cells. These cells are used to encode the respective recombinant segments. Segment encoding a nucleic acid binding protein that enhances vector uptake Enrich your account. The recombinant segment from the cell expressing the marker is then Can be subjected to further rounds of selection. Usually, the recombinant segment is first , Recovered from cells (by PCR amplification). The recombinant segments are then Or recombination with other sources of DNA-binding protein variants for further recombination A segment may be created. Additional recombinant segments are screened in the same manner as before. Lean.   In a modification of the above procedure, the binding site recognized by the DNA binding protein However, instead of or in addition to DNA binding proteins, obtain. The DNA binding site is such that the starting substrate contains a variant binding site and these The recombination form of the site is similar to the components of the vector encoding the DNA binding protein Similar to DNA binding proteins, except that Evolved.   Evolved nucleic acid segments encoding DNA binding proteins, and / or evolved The designated DNA binding site can be included in a gene therapy vector. DNA binding protein If the affinity is specific for a known DNA binding site, the binding site and D A sequence encoding an NA binding protein can be inserted into a gene therapy vector for gene therapy. Include with such coding and regulatory sequences as necessary to effect Is enough. In some cases, the evolved DNA binding protein is Vector that may not have a strong sequence specificity and used for screening It may be unknown exactly which site on the protein is bound by the protein. This In these situations, gene therapy vectors may require all or most screens. Vector sequences with additional sequences required to effect gene therapy Should be included in   An exemplary selection scheme is shown in FIG. The lower left part of the figure shows the two vectors , Each have the same marker and DNA binding site, and the vector Differs in the recombinant segment encoding the protein. The vector is E. coli. col Transfected into i-cells. Vectors are expressed in cells, Produce different proteins that differ between different cells. Recombinant binding proteins Complex with the vector encoding it, and these complexes are Is stored in The complex is then contacted with the eukaryote of the recipient. Eukaryotic object Has several different cell surface receptors, one of which is a DNA binding protein. It can interact with one of the qualities to facilitate DNA uptake. Selection Ma on Vector Selection for expression of the protein identified cells transformed with the vector. You. These cells are subject to recombinant segments that provide enhanced DNA uptake. Enriched.   (J) Improved intracellular stability of vectors   With larger and improved cell maintenance, intracellular stability, and expression characteristics Such vectors can be evolved using the recursive recombination method of the invention. Many genetics In child therapy, the vector is stably maintained in the target cell and To allow infinite expression. This is because viruses and non-wi This is true in both cases of the Lus vector. Vector progress towards improved maintenance Substrates for transformation and recombinant formats can be selected according to the principles described above. Substrate If it is a fragment of the vector genome, the recombinant product is Reinserted into the vector genome. Vector genomes are A selection marker that replaces or is fused to a therapeutic coding sequence carried by the vector. May often be contained. For screening, the vector genome is If it does not already exist as a result of in vivo recombination, The vector genome having the vector is introduced into a cell. Cells can be grown without selection markers A number of passages are propagated, thereby reflecting the in vivo situation. Where the typical It is not possible to make a choice for maintenance of the therapeutic gene. Increase the appropriate period After culturing, a selection for the marker is applied and the surviving cells are harvested. This These cells have recombinant segments (which provide improved maintenance properties in the cells) That is, it may contain a vector having a stably maintained recombinant segment). . In some cases, at least partially improved maintenance properties, cellular genomics Resulting in improved and more stable incorporation into the system. Improved replication, fiber Recombinant segments with retention and / or stability properties are recovered from cells. And new with each other and / or to generate additional recombination segments Either with the fresh substrate, it is subjected to a further round of recombination. These are: Screened in the same manner as the previous recombinant segment.(K) Reduced immunogenicity of the vector   Protein and nucleic acid sequences with reduced immunogenicity It can be evolved using permutations. Immunogenicity should be considered especially with viral vectors. It is. Because the host immune response (including CTL-mediated and humoral responses) Make the virus reach its intended target, especially in repeated administrations Because it can be prevented. Prevent the virus from reaching its intended target Cellular immune responses can also be in naked form or coated on liposome-like coats. Can be directed against a non-viral vector administered in a native fashion.   The host's immune response to eliminate infected cells is also a major factor in gene therapy. It is a problem. Although CTL is primarily responsible for eliminating infected cells, the problem is also It can be targeted or completely antibody mediated. The recursive recombination method of the present invention To reduce this (especially cellular) immunity to infected cells, Can be used to modify In a modification of this embodiment, the adenovirus For mediated gene transfer, adenovirus late gene expression is Induced by the method of the present invention to reduce the CTL response that contributes to cell elimination Reduced mutations. Therefore, it is seen in adenovirus-mediated gene transfer. The problem of transient maintenance of the resulting virus is reduced.   Substrates and formats for recombination generally follow the principles described above. Generally, the outer surface Regions of the viral genome that encode proteins head for reduced immunogenicity Provides the most likely starting substrate for evolution. Alternatively, the vector genome The whole can be included as a starting substrate for recombination. The recombinant virus genome Should be packaged as a virus before screening, and non- The viral genome can be used in the proposed compositions (eg, liposomes) for therapeutic administration. )).   Viruses containing properly formulated recombinant or non-viral genomes , Mammals (eg, mice, rats, rabbits, pigs, horses, primates, or G) and live virus or non-viral genome is Recovered after septum. Often, the administration is intravenous, and the live virus and And non-viral genomes are recovered from blood. Viable and non-viral Noms are enriched for recombinant segments that confer reduced immunogenic properties You. These recombination segments will allow some of the substrates or Is used as all. Subsequent selections follow the same format.   In a modification of the above format, the antibody is immunized with a viral library. Recovered from dairy animals and immobilized on columns. Next, the virus library Or another aliquot of a derivative library resulting from further rounds of recombination Can be applied to the column and the virus that has passed through the column can be recovered. this These viruses are enriched for viruses with low immunogenicity.   In a modification of this method for non-viral vectors, the therapeutic development of the vector The current product is linked to a DNA binding protein that has an affinity for the sequence on the vector. Expressed as a ligated fusion protein. Thus, at least some Is maintained physically close to the vector that produces it. Therefore, An immune response to the expression product also removes the vector sequence. Therefore, in animals By recovering vector sequences that survive for a certain period of time, vectors having low immunogenicity per se Enrich both the promoter sequence and the sequence encoding the expression product with low immunogenicity You.   (L) Reduced toxicity of vectors   Protein and nucleic acid sequences having reduced cytotoxicity are described in the recursive set of the invention. It can be evolved using permutations. Toxicity caused by the expression of viral genes There are sometimes concerns when using viral vectors for gene therapy. The present invention The method uses mutations that block viral DNA replication and gene expression in vivo. Can be used to derive and select multiple combinations of In vitro To produce the activated virus, these mutations are Temperature sensitive or nonsense mutations so that they can be grown in vitro under Should be a conditional mutation. The multiplicity and therefore the degeneracy of the conditional mutation is Prevents the variant virus from reverting to the wild-type genotype or phenotype.   (M) Improved specificity of integration   Viral sequences with improved specificity of integration can be used in the recursive recombination of the invention. It can be evolved using law. For example, AAV is preferentially located at a site in chromosome 19q13.3. It is known to be incorporated. Integration at this site is exogenous at this site Presence of DNA sequence has no deleterious effect on endogenous cellular gene expression This is advantageous. Therefore, it may increase the specificity of AAV for this site It is desirable.   The starting material for the recombination should be at least the ITR and, if necessary, the rep gene. AAV vectors to include. Because the latter, in site-specific recombination, It can have a role. Public role in site-specific integration Genes from other viruses that are known or thought to have May be included. Preferably, the genome contains a marker sequence. Recombination is Produce a library of AAV viruses with different viral segments in the system Proceeds through any of the previously discussed recombination formats. AAV virus Used to infect appropriate target cells. Cells that have taken up AAV DNA , Can be recognized from the expression of the marker. Genomic DNA is isolated from these cells And the central region of the intended site of integration is amplified by PCR. The amplified region is transformed into a recombination segment that confers the desired properties of site-specific recombination. About being enriched. These recombination segments start for the next round of recombination Form the material.   Similar principles apply to other viral vectors, and indeed non-viral sequences and vectors. Applied to the operator. For example, as described above, one embodiment of the present invention Using a specific integration system, the recombinant sequence of interest is ligated to a specific, constant Target. Preferred embodiments include Cre / LoxP or related FLP / FRT site features. Use a heterogeneous integration system. Cre / LoxP system uses Cre recombinase enzyme 34, site-specific insertion and specific palindromes of viral or phage vectors. Mediates excision into base pair sequences ("LoxP sites"). Recursive sequence recombination method of the present invention Can be used to modify these systems (eg, to improve the specificity of integration, Modification of recombinase activity to create alternative specific sites for integration For example).   In a further embodiment, the starting vector has any preferred integration sites. It is not necessary to do so. In this case, do not interfere with the expression of other genes. Appropriate chromosomal sites are selected for subsequent recombination and selection. Cycle until the vector has evolved to preferentially integrate at that site. Will be(N) improved resistance to microorganisms   The recursive recombination method of the present invention is a novel inhibitor of microbial and viral infections Known inhibitors of microbial and viral infections (microorganisms) And trans-dominant inhibitors of product and viral replication and gene expression) Can be used to improve In some gene therapy applications, vectors May encode a product that is an inhibitor to a microorganism such as a virus. Due to the complexity of the virus life cycle and the inherent variability of the virus, Recursive sequence recombination may have improved potency and / or novel specificity or improved Practical tools for evolving protective antiviral constructs with improved specificity It is. This can be achieved using any of a variety of mechanisms. For example, gene therapy Therapeutic vectors use anti-sense to block expression of the mRNA of a virus or other pathogen. RNA can be encoded. Antisense RNA contains important regulatory sequences (eg, promoters). Or to a coding sequence, or both. You. Alternatively, the vector may be a tag that inhibits pathogen replication or gene expression. Can code for protein. For example, many gene therapy strategies include mature T cells. It is designed to inhibit HIV-1 replication in cells. T cells are blood lymphocytes The insertion of antiviral genes into hematopoietic stem cells Acts as a vehicle that provides long-term protection in committed stem cell-derived primordial T cells I do. One such “cell immunization” strategy is the HIV-1 rev trans The gene encoding the sex mutant protein RevM10 is used. RevM 10 is a T cell Has been demonstrated to inhibit HIV-1 replication in strains and primary T cells; Bonyh adi (1997) J. Virol. 71: 4707-4716; Nabel (1996) Gene Therapy, abstract 361 , CSH. HIV-1 tat and rev mutants also have the potential for HIV-1 replication. It has been suggested to be a trans-dominant inhibitor in the vesicle (Caputo (1997) Ge ne Ther. 4: 288-295). Another candidate for evolution according to the method of the invention is trans An active transcriptional regulatory protein, IκBα, which regulates its effect on HIV transcription. Creates a cellular inhibitor of human retrovirus replication by an independent mechanism (Wu (1995) Proc. Natl. Acad. Sci. USA 92: 1480-1484. ). Repeated inhibitors from viral fragments (eg, poly TAR constructs ) Can also be used as inhibitors of HIV-1 gene expression. TAR, HIV remains RNA stem loop bound by activators or inhibitors of gene expression Structure. TAR mediates (eg, saturates) cellular factor / RNA interactions And this means that the activator of transcription (eg, Tat) action can be Suggests that such competing TAR reactions in vivo may be inhibited Baker (1994) Nucleic Acids Res. See 22: 3365-3372. Times of the present invention Recursive recombination methods can evolve and improve these and related intracellular inhibition systems.   Proteins from one virus or virus product are blocked from other outbreaks There are also many examples that can be harmful. Woffendin (1994) Proc. Natl. Acad . Sci. USA 91: 11581-11585. In particular, at least from adeno-associated virus (AAV) One protein is known to be inhibitory to HIV. AAV big The rep gene products (Rep78 and Rep68) are used to track AAV DNA replication and AAV gene expression. It is a pleiotropic effector protein that is required for regulation of genes. These essential Apart from their functions, these rep products are responsible for the replication and replication of HIV-1 and many DNA viruses. And may inhibit gene expression. Batchu (1995) FEBS Lett. 367: 267-271; Antoni (1991) J.C. Virol. 65: 396-404. The recursive recombination method of the present invention A new interstitial inhibitory protein can be developed and improved.   The present invention improves the quality of inhibition of antisense RNA and the above proteins. And also provide a means for identifying novel inhibitors. Known inhibitors Improvements to several aspects (eg, improved expression, improved stability, or Or slightly altered binding specificity). In the method of the present invention Need not know which of these contributing properties is being improved; Is about the ultimately desired property of microbial resistance.   Due to the development of known inhibitors, substrates for recombinant and recombinant formats are described above. Choose according to principle. The substrate may be a viral vector genome encoding the inhibitor. Or a portion thereof, and related regulatory sequences. Early in recombinant substrates Diversity can be natural or induced. After the round of recombination, (If they are not already present in the cell as a result of in vivo recombination), The cells are then contacted with the microorganism whose protection is desired. Exposure to microorganisms Cells that survive the enrichment for recombinant segments that confer resistance to microorganisms Become These recombination segments serve as substrates for the next round of recombination. Form some or all.   A similar principle applies to novel inhibitors of inhibitors that should be expressed from gene therapy vectors. It can be applied to identification. More rounds of recombination and screening are May be needed to get a reasonable result. For example, the virus to be inhibited or Sequences encoding viral proteins from other viruses are suitable for recombination. Provides a clear initial substrate. The coding sequence was obtained from the same or a different virus. And the natural diversity can be increased by inducing additional mutations (eg, It can be increased as described above (by error-prone PCR). Recombination and screenini Ringing is also performed as described above.   In an exemplary embodiment, the library of variants is a candidate construct (single or single). Are constructed on the basis of a plurality of (the examples are described above). The library is targeted The cells are transduced or transfected. Attack cells with the desired microorganism . For example, survival against cell necrotic virus, or Based on the current lack, resistant cells are isolated. It is an inserted mask such as GFP. Protein gene. Using these methods, viral replication or gene Detect cells where the current is blocked. FACS or viral code surface epitome Panning with antibodies against the surface or induced surface epitopes in the positive selection step To use. The gene encoding the resistance factor is recovered, for example, by PCR. Until the desired level of protection against microorganisms is achieved, the recovered genes are Such additional rounds of recursive sequence recombination may be subjected.   Further illustrative examples of antiviral mechanisms that can be improved by the methods of the invention include: Includes antiviral ribozyme system. For example, one or more ribozymes may be Can be targeted to A. Adenovirus delivers anti-hepatitis C ribozyme Lieber (1996) J. Am. Virol. 70: 8782-8791; Ohkawa (199 7) J. Hepatol. See 27: 78-84. HIV-1 Rev response element (RRE) region specific Hammerhead ribozyme completely inhibits HIV-1 replication (Duran (1997) Ge See ne Ther 4: 533-543. Sendai virus polycistronic P / C mRNA Can be cleaved by ribozymes; Gavin (1997) J. Am. Blol. Chem. 272: 1461 -1472.   Antiviral cytokines can also be improved by the methods of the present invention. For example , Wild-type or chimeric of wild-type interferon (eg, IFNα17 construct, IFN β construct, and IFNγ construct) can be subjected to recursive sequence recombination. these Sequence can be placed under the control of a viral activation promoter (e.g., the HIV mini LTR). See Mehtali (1996) Gene Therapy, abstract # 364, CSH. For example, Stable IFN transgene under HIV-1 Tat protein positive control Cell lines that are highly resistant to HIV-1 replication in vitro. This anti Virus resistance is associated with a strong induction of IFN synthesis immediately after viral infection. But I FN-γ-transfected cells have a high level of induction of IFN-γ secretion despite induction. Enabled HIV-1 infection in vivo (see Sanhadji (1997) AIDS 11: 977-986). thing). Using the methods of the present invention, anti-c Ilus can be developed.   Single Chain or Fab Directed Intracellularly to a Viral Component Using the Methods of the Invention Antibody fragments can be developed; Marasco (1996) Gene Therapy, abstract 160 , CSH. For example, in somatic gene therapy to treat HIV-1 infection One strategy for this is intracellular expression of anti-HIV-1 Rev single chain variable fragment (Sfv) Duan (1997) Gene Ther, supra. HIV integrase catalytic domain and Intracellular expression of the carboxy-terminal domain produces productive resistance to HIV-1 infection I will. This inhibition of HIV-1 replication is localized to either the cytoplasmic or nuclear compartment of the cell. Observed for localized Sfv. Levy-Mintz (1996) Virol. 70: 8821-8832 checking ... Expression of anti-reverse transcriptase (RT) Sfv in T lymphocytes is integrated In the previous step, it specifically neutralized the RT activity, and the reverse transcription process (HIV-1 line) Early cycle events). Virus at these early stages Locking affects HIV-1 proliferation and HIV-1 induction in susceptible human T lymphocytes. Cellular necrotic effects are dramatically reduced by preventing the formation of proviral DNA Reduce. Shaheen (1996) J.C. Virol. See 70: 3392-3400. More The described method describes the potency and range of such anti-viral intracellular antibody fragments. Enclosure can develop.   Directed to improved virus binding aptamers or virus components, as described above Identified peptide ligands can also be further developed by the methods of the present invention. An example For example, an RNA aptamer that recognizes a peptide fragment of human HIV-1 Rev Binds to free peptides more tightly than RNA ligands (Rev binding elements) (Xu (1996) Proc. Natl. Acad. Sci. USA 93: 7475-7480; Symensm). a (1996) J. See Virol 70: 179-187. Isolated from single-stranded DNA preparations The aptamer sequence has thrombin inhibitory activity, which is It is believed that tamamers are present in the mammalian genome and can build an endogenous antithrombin system. And Similarly, using the recursive sequencing method of the present invention, as an antimicrobial agent, and Generally further identifies, develops, and develops useful aptamer sequences for gene therapy. Can be improved.   (O) Virus packaging cell line   The recursive sequence recombination method of the present invention also provides a new and improved virus package. Can be used to develop zing cell lines. C used in gene therapy The ills vector is usually packaged into viral particles by the packaging cell line. Caging. Typically, the vector is transferred to the packaging and subsequent host. Contains the minimal viral sequences required for the integration of , Are replaced by an expression cassette for the protein to be expressed. Missing c The ils function is provided in trans by a packaging cell line. For example, AAV vectors used in gene therapy are typically packaged and packaged. And only the AAV genome-derived ITR sequences required for integration into the host genome. Have. Package the viral DNA into a cell line. This cell line has been Contains helper plasmids encoding genes (i.e., rep and cap) Lacks an array. The cell line is also infected with adenovirus as a helper. Hell Parvirus promotes AAV vector replication and is derived from helper plasmid A Promotes expression of AV genes. Helper plasmids have been Not packaged in significant quantities. Contaminants related to adenovirus include, for example, Adenovirus can be reduced by heat treatment, which is more sensitive than AAV. AAV recombination The body is generally produced by a transient co-transfection method. why If it turns out it is difficult to generate a stable packaging cell line (Maxwell (1997) J. Virol. Methods 63: 129-136).   The goal in improving a packaging cell line is to use a stable packaging cell line. Producing; increasing the yield of the packaged AAV vector; Reducing the ratio of AAV progeny to Ruper virus; and packaging Reduce the toxicity of the rep gene to cells, which in turn can lead to higher yields of AAV. And Leading candidate genes for development / modification according to the method of the invention are: AAV replication (rep) and capsid (cap) genes, which are AAV helper plasmids. May exist on the network. Overexpression of the rep gene can reduce AAV DNA replication, and Cap gene expression can be strongly inhibited, and reduced rep levels indicate that cap gene expression And supports normal rAAV DNA replication. Therefore, the regression of the rep gene Recombinant modifications and their expression can result in increased AAV vector production (Li (1 997) J.C. Virol. 71: 5236-5243).   These and related sequences are regressed according to the general principles discussed. Subject to specific sequence recombination. That is, mutant forms of these genes can be Or a recombinant segment resulting from recombination, either in vitro or in vitro The cells comprising the desired characteristics (eg, a stable packaging cell line; Yield of packaged AAV; increased viability of cells; or packaged AAV (Low yield of herpes virus as compared to). the same Applying the principle, simultaneously or simultaneously with the development of the AAV gene on the helper plasmid Subsequently, the gene in the helper adenovirus may be developed either.   The cellular genes in the packaging cell line that affect packaging are also It can be developed without knowing what these genes are. This means that Achieved by transforming the caging cell line with a library of genes, Some of the libraries recombine with cognate genes in the packaging cell line I do. The library of genes can be obtained from another type or species of cells, Or a mixture of several types and species and / or Can have diversity induced by processes such as PCR. Recombinant gene Containing cells with improved packaging characteristics (eg, increased yield of AAV virus). Amount). If necessary, additional libraries The screens from the previous round can be transformed into surviving cells. is there Alternatively, the pool of surviving cells is split in two, and the DNA is isolated from one half and Can be used to transform the other half. In this way, the first round The best recombinant segment identified in the screening of In the recombination of the und, recombination with each other is performed.                                  Example Example 1: M13 scFv library   This example demonstrates that sc for localization to a given cell type (eg, xenogeneic neoplasm) Figure 4 shows in vivo panning of a library of bacteriophages displaying Fv. sc Fv Antibody-Phage Display Library for Crameri (1996) Nature Medicine 2: 100-10 Constructed as described in 2. E. coli in LB containing 50 μg / ml kanamycin. coli T After growth of the phage library on G1, bacterial cells are removed by centrifugation, The phage were then added with up to 4% PEG and NaCl to a final concentration of 0.5M. Precipitated by addition. After 1 hour incubation on ice, the solution was Centrifuge at xg for 30 minutes and pellet the pellet with Dulbecco's phosphate buffered saline. Resuspended in water (DPBS).   Male Sprague-Dawley rats are anesthetized and phage is injected intravenously and blood Fluid was sampled from the artery via the ipsilateral femoral artery catheter. EDTA blood Used in samples to reduce clotting. Blood samples were taken immediately after phage administration. Before and 7.6 × 10¹¹5, 30, 60, 120, and 24 after injection of colony forming units Collected at 0 minutes. Phage titers were determined by dilution of whole blood in DPBS and E. coli. coli TG1 infection And M13 colony forming units were assayed. 4 iterations of the protocol Was carried out. M13 bacteriophage showed 6 in rat blood after in vivo injection. Is stable with a half-life of hours and remains infectious (against E. coli). I found that.Example 2: Panning of M13 scFv library for specific localization   The scFv antibody phage display library was cloned into a To mice. After an appropriate incubation time, the tumor tissue is collected, and Phage are eluted from the collected tissue by homogenization of the tissue sample.   Aliquots of the recovered phage were administered for at least one additional cycle, and And in vivo selection by the same protocol.   Purify the DNA using an aliquot of the recovered phage and filter the recovered DNA Recursive recombination by shuffling in vivo, and shuffling The resulting population of M13 genome introduced into E. coli and packaged; Recover the shuffled library of M13 species and Administer mice to mice for vehicle administration and in vivo selection according to the same protocol .   An aliquot of the recovered phage was used to infect E. coli at multiple infections. E. coli, The M13 genome is recursively recombined in vivo by shuffling and shuffled. The resulting population of fling M13 genomes was into E. coli and package Recovering the mixed library of M13 species and at least one additional Cycle administration and administration to mice for in vivo selection according to the same protocol I do.Example 3: MGMT gene evolution   This example demonstrates improved properties for protection of human bone marrow against alkylating drugs. The following illustrates the evolution of the MGMT gene. Wild-type human MGMT on a high copy number plasmid  The cDNA was amplified by PCR and fragmented randomly with DNase. Small fragment Fragment (50-100 bp) with Taq DNA polymerase to size the starting fragment. In the process of inducing point mutations at a proportional rate, Reassembled into long fragments (Stemmer (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751). All genes encoding 207 amino acids were This is a measure of all proteins that contain a functionally important DNA binding domain. Mutagenesis in all regions (Kanugula (1995) Biochemistry 34: 7113-7119). ). The full-length fragment is cloned into a vector and Ferase deficient E. coli (GWR111 strain, ada ogt) (Rebeck (1991) J. Bacteriol. 173: 2068-2076). Create relatively large number of mutants and increase diversity did. This is because inactivating mutants are not This is because it can be removed by child selection. In this selection, bacteria are treated with the methylating agent MNNG three times in a row. Each of which is separated by a one-hour collection time. Let the bacteria make more MGMT in the meantime. The triple selection is for cells with inactivated MGMT. Proteins that kill vesicles and have improved expression and / or activity of MGMT Give priority to quality.   The improved human MGMT gene also contains both native and unnatural coding sequence diversity. Produced by using sex. Unnatural diversity is created by random fragmentation of human MGMT (Wild-type MGMT cDNA was obtained from Dr. S. Mitra (University of Texas, Galveston) Courtesy; cDNA and protein sequences, and residues For numbering, see Tano (1990) “Human DNA repair for 06-alkylguanines”. Isolation and Structural Characterization of a cDNA Clone Encoding the Recombinant Protein "Proc. Natl. Acad. Sci. USA 87: 686-690). After this, the mutagenized DNA shufflin Reassembly of the fragments in the reaction follows. Alkyl transferer Active alterations selected for their ability to confer MNNG resistance to zeo-deficient E. coli Pool variants, remutate, and rejoin in subsequent cycles of shuffling The combined (alkyl transferase deficient (ada ogt) E. coli GWR111 strain is L. Sam. son, Harvard University, Cambridge, MA; Rebeck (1991) J. Bacteriol. 173: 206 8-2076). Using two cycles of simple DNA shuffling Built, non-natural diversity.   Wild-type human alkyltransferase (MGMT) cDNA was transferred to pUC118 plasmid (N ew England Biolabs, Beverly, MA) and encoding nucleotide residue number 380. Was subcloned into the translationally silent XhoI site created in. Adjacent Non-coding sequences are removed from the construct and coli ribosome binding site Positions are added via PCR amplification using oligos 1 and 2 (see below), Then, it was inserted into the EcoRI-HinDIII site of pUC118.   This construct is named "pFC14". The sequence of all MGMT genes in pFC14 was Was verified as its ability to complement was verified. A non-functional dummy vector, Active site coding region between the XhoI site and the PinAI site (nucleotide residues no. 521 (Tano (1990) supra, for residue numbering)) and oligos 3 and 4 (hereinafter A) synthetic stuffer made by annealing Constructed by replacing with a strand. The inactivity of this gene was verified by its ability to not complement GWR111. Short form The dummy vector from which MGMT has been removed is used as a cloning vector for library construction. Used to reduce the potential for contamination by wild-type MGMT.   Live gene randomized by random fragmentation and reassembly The general procedure for making rallies is described in Stemmer (1994) Proc. Natl. Acad. Sci . USA 91: 10747-10751; and in Stemmer (1994) Nature 370: 389-391. Used as posted. The starting material was a 1.2 kbp (kilobase) made from pFC14. Spare) Oligo # 5: 5'-AAGAGCGCCCAATA, PCR product and outer primer CGCAAA-3 'and oligo # 6: 5'-TAGCGGTCACGCTGCGCGTAA-3', and Taq DNA Produced using Limerase (Promega). This product is located adjacent to human MGMT + pFC14. Columns, then Stemmer (1994) Proc. Natl. Acad. Sci. USA, before And Stemmer (1994) Nature, supra. Were adjusted and reassembled with Taq DNA polymerase. Nesty # 7: 5'-ATGCAGCTGGCACGACAGGTTT-3 'and oligo # 8: Re-amplification using 5'-TACAGGGCGCGTACTATGGTT-3 'was performed with EcoRI and HinDIII. A 980 bp fragment was obtained. The resulting 650 bp fragment was Ligation into a dummy vector. Electroporate the ligation mixture into GWR111 And about 10 per cycle^FiveOf active clones Selected. Select the inducer isopropyl-β-thiogalactopyrano Except for the abbreviation of Sid, Christians (1996) Proc. Natl. Acad. Sci. USA 93: 6124 Performed as described in -6128. The bacteria in the culture were treated with three successive doses of MNNG. , Each separated by a one hour recovery time. After the third dose, pre-load all cells. Spread on the board. The next day, colonies are pooled and MGMT for the next cycle  DNA was prepared by PCR using oligos # 5 and # 6 (above). This step A total of 6 cycles were repeated. MNNG processing, as shuffling progresses Start with 3 × 10 ug / ml MNNG progressively more stringent To reach 50 ug / ml. Similarly, less colonies later Caught for shuffling in half cycle.   Four known mammalian alkyltransferases (rat, mouse, hams Improved human MGMT genetics, also using the natural diversity of Sequence diversity in offspring was generated. As shown in FIG. Sequence alignments show regions of extensive homology as well as regions of diversity. 2 × 10²⁸Of known natural amino acid substitutions from mammalian alkyltransferases Combinations exist (52 positions with 2 amino acids, 24 positions with 3 amino acids , And 2 positions with 4 amino acids = 2⁵²× 3^{twenty four}× 4^Two). This diversity has been These oligos were used at equal ratios used throughout the use of the oligonucleotides (FIG. 3). Mixed together, the DNA fragments during the reassociation reaction in the third and fourth cycles One diverse pool was created that was mixed with Oligos with several different molar ratios : Fragments are generated and high concentrations of oligonucleotides inhibit reassociation Observed that. Probably because many base pair mismatches Because it is overwhelming. Those mixtures that produce proper reassociation are correct after re-amplification. The highest ratio of oligos, one orientation, is determined by the size of the new product. A mixture containing a molar ratio of ゴ: 4 fragments was selected for further cycling. Was. Annealing of each oligonucleotide to the human-derived MGMT sequence is degenerate or Enabled by the homology of 20 nucleotides to both sides adjacent to the non-human sequence. Was. Control PCR shows that all oligonucleotides are approximately equal to the human sequence It was shown that hybridization was possible.   In the third shuffling, the oligonucleotide was referred to as "non-naturally occurring". "Like", ie having pooled human MGMT clones surviving in cycle 2 The oligonucleotide was combined with the generated sequence. Conditions are meeting Incorporate oligonucleotides and maintain diversity while maintaining the correct size of objects Changed in an attempt to maximize gender. To ensure accurate meetings The maximum molar ratio of lignonucleotide: fragment was 1: 4. Ratio system Due to limitations, "oligo spiking" was repeated in cycle 4. Follow Thus, the pools in cycles 3 and 4 contain randomly mutated human sequences. And hybrids containing separate combinations of mammalian MGMT gene segments. Was. These pools were subjected to selection between cycles. More Oligonu Two final rounds of "normal shuffling" without cleides (Cycle 5 and And cycle 6) in an attempt to further evolve the hybrid protein Carried out.   Individual clones surviving in later cycles were isolated with a single 40 ug / ml dose of MNNG. For improvement by processing them and comparing residuals with untreated samples Screened. The best clone from cycle 4 is wild at this dose It showed an improvement over 10 times the mold. The estimated data shown in FIG. 5 (SEQ ID NO: 2) The protein (amino acid) sequence is 7 amino acids from wild-type human alkyltransferase. Amino acid differences (see seven circled amino acid residues in FIG. 5), other mammals 5 residues found in animal alkyltransferase (circled in FIG. 4) Based on the improved (evolved) nucleotide sequence (SEQ ID NO: 1) Follow. These five amino acid changes were due to the spikes during cycles 3 and 4. It appears to be encoded by a lignonucleotide. All five have the same degenerate orientation Gonucleotide pool (# 7 in FIG. 3). Other amino acid changes (Q (gln) to R (arg) (Q72R) at residue number 72, G (gly) to D (as p) (G173D)) (Tano (1990) supra) is not shown in natural diversity and therefore Produced by the shuffling process. In addition, 2 (from wild type) Two translationally silent nucleotide changes were detected (two lower in FIG. 5). See lined nucleic acid residues).   This shuffling mutant is more thorough about its activity in E. coli Characterized. In one set of experiments, cells were treated with graded doses of MNNG, and The surviving fraction was determined. Plus isolated from individual clones surviving MNNG treatment The mid was transformed into GWR111. Retransformed clones can be used at a single 40 ug / ml dose. Were screened separately by treating them with MNNG. The best Clones were further analyzed in three ways: (i) the entire MGMT DNA sequence was Dye terminator cycle sequencing (Applied Biosystems 373A Autosequenc er, Foster City, CA). (Ii) the killing curve was plotted against the absence of isopropyl-β-thiogalactopyranoside. Numerically growing cells are treated with a graded single dose of MNNG and untreated control Established by measuring the ability to form colonies for trawl. Wild type inheritance (PFC14) or V139F mutant (Cristians (1996) Prod. Natl. Acad. Sci. USA, supra) ) Were processed in parallel for comparison; (iii) the bacterial extract Transferase activity described in Bobola (1995) Molec. Carcinogen 13: 70-80. As in O⁶− [^ThreeIn vitro exposure to bovine thymus DNA containing [H] methylguanine Quantified. Some extracts are used for mammalian alkyl transfer -Zein inhibitor O⁶-Preincubation with benzylguanine.   Survivors are larger than cells with either wild-type human MGMT or the V139F variant. It was terrible. LD_TenThe dose of MNNG giving 10% survival is wild type, 17.5 ug / ml V139F, 25ug / ml; and cycle 4 shuffling mutant, 33ug / ml; Was. In a second set of experiments, bacterial extracts were washed with excess [^ThreeH] methylated DNA substrates (primarily and O⁶-In the form of methylguanine) in vitro Transferase activity was measured. Per minute per ug of total protein Mean insoluble counts were: wild type, 126; V139F, 58; and cycle 4 shuffle Mutants, 52. All three proteins are inhibitor O⁶-Benzyl Sensitive to guanine.   Thus, the recursive sequence recombination method of the present invention provides a new and improved human alkyl Transferase protein was successfully produced. Mutation shuffling process Random diversity generated by natural resources is due to the diversity provided by nature. Was increased. The natural diversity is to transform human gene fragments into known mammalian Simply shuffle oligonucleotides encoding all of the Can be used. Human genes in sequences flanking regions of diversity Homology facilitated oligonucleotide incorporation. The best mutation The body contains a human alkyltransferase, as shown in FIG. 5 (SEQ ID NO: 1). Was a hybrid having a difference of 7 amino acids from Two of the variants are Occurs naturally during shuffling, and the other five reflect natural diversity, especially Encoded by one of the "spike oligos" cross-linked at no acid position 50. all Was shown to be able to hybridize to human sequences by PCR. , All seem to have been incorporated into the pool, at least to some extent.   Previous work with different systems also showed that synthetic oligos in such reactions It was confirmed that it would be incorporated at almost the expected ratio (Crameri (1996) Nature Me dicine, supra). Another way to incorporate natural diversity is to use cDNA from various sources. By isolating or synthesizing and shuffling all coding sequences together is there. This recursive method of propagating natural diversity is different for different organisms and organisms within organisms. Improve many related genes from the gene family. Furthermore, it has the structure Apply to multiple proteins with motifs related either functionally or functionally Can be done.   How Amino Acid Substitutions in Shuffling Mutants Are Not Possible in E. coli It is difficult to rationalize mechanically to increase activity. The only alkyl tiger Crystallographic structure of the bacterial Ada protein C-terminal fragment (Wibley (19 95) Based on Cancer Drug Design 10: 75-95; Moore (1994) EMBO J.13: 1495-1501)) Of human alkyltransferase in computer models No amino acid positions were mutated in the shuffling mutants defined. Rank Clustering of the five mutations around position 50 is significant, but this region of the protein There was no known function due to. Three of these five substitutions are It has been found in other mammalian alkyltransferases. Several The substitution can be a neutral possibility that can be answered by backcrossing, while the other May be synergistic with substitutions, particularly involving potential changes. From G (gly) at position number 173 Mutation to D (asp) (G173D) (see Tano (1990) supra for numbering of residues) Approximation with conservative E (glu) (E172) at residue number 172 There may be significant predefined proposed involvement in salt-related interactions. In this area Additional acidic residues in the form can enhance this effect.   The power of DNA shuffling can be attributed to many such duplications that should not work in detail. A molecular growth process that allows for a combination of mutations that progressively improves is there. We pioneered this property and found that natural enzymes or any reported Evolve more potent alkyltransferases in vivo than mutants. This progress The mutated variants are very useful for chemoprotection by gene therapy. Wild type Improvements over alkyltransferases are not consistent with patient toxicity. It is very useful to clinicians by allowing for increased dosages of the ruquilizing agent. Once an alkylating agent that is not used due to severe bone marrow toxicity is expected, May be acceptable. Low levels of alkyltransferases in vivo Even improvements can enable relatively few resistant cells to repopulate the bone marrow Useful for giving a selection. This alkyltransferase is further modified O⁶-Incorporate additional features such as benzylguanine resistance. this The alkyltransferase can also be subjected to further DNA shuffling, And improved nuclear localization, or better interaction with eukaryotic chromatin structures Can be selected for further improved activity in mammalian cells such as .Example 4: In vitro recombination using adenovirus-phagemid Russ whole genome shuffling   This example packages DNA inserts over 10 kilobases in size. Of a novel adenovirus phagemid using the recursive recombination method of the present invention 1 shows the construct. Incorporation of the phage f1 origin using the methods of the invention also New capable of evolving the entire genome of viruses such as the 36 kb family of adenovirus Create a standard in vivo shuffling format.   The widely used human adenovirus type 5 (Ad5) has a genome size of 36 kb. I do. Creating an excessive number of changes that can result in a high percentage of non-manufactured recombinant variants In addition, shuffling this large genome in vitro is difficult. To minimize this problem, and achieve Ad5 whole-genome shuffling Adenovirus-phagemid was constructed using the method of the present invention.   As outlined in FIG. 6, the 36 kb Ad5 genome was cut into two overlapping regions by restriction digestion. Divided into minutes. Each of the two halves was subcloned into pBR322; obtained The two plasmids were called pAd-R and p-AdL. For more information, Adapters were first ligated to each end of the linear 36 kb genomic DNA. Then This product was digested with BamHI and the right half of the Ad5 genome (nucleotides 21,562- 35,935); and cut with EcoRI to the left half of the genome (nucleotides 1-27). , 331). Then, the right half of the 14.3 kb BamHI / EcoRI fragment was Ligation with coRI digested pBR322 to create Ad-R, and the 27.3 kb EcoRI fragment pAd-L was created by ligation with oRI digested pBR322. Gene transfer and safety reasons For this, the Ad5 E1 region was then: first used by site-directed mutagenesis (G residue at position 457). By changing the group to a T residue and the C residue at position 459 to an A residue) Create an AflII site at Otide 455; then another AflII site is present in the plasmid Therefore, it was deleted from pAd-L by performing AflII partial digestion. 24.3kd  AflII fragment is gel purified and filled in with DNA polymerase I to create blunt ends did. It was then ligated with a SwaI linker to create an E between nucleotides 458 and 3533. 1 region at the same time and a unique SwaI site for insertion of the foreign gene. Inserted.   To construct a phagemid, the ssDNA phage f1 origin of replication was II Obtained by PCR from KS (-) phagemid (Stratagene, San Diego, CA) and As illustrated in FIG. 6, the Ad plasmid (pAD-R and And pAD-L-1) to the ClaI site. Next, the obtained Ad phagemid was mutD5 (see Degnen (1974) J. Bacteriol. 117: 477-487) Gained variants, thereby increasing diversity. The natural mutation rate of mutD5 is about 1.8 × 10^-6 / Base pairs / cell / generation (FiJalkowska (1996) Proc. Natl. Aca. Sci. USA 93: 2856-2861), which is about 10% higher than the in vitro shuffling mutation rate. 0-fold lower (see Stemmer (1994) Proc. Natl. Aca. Sci. USA 93: 2856-2861).   To prepare phagemid phage, these mutated Ad phagemids Was purified from mutD5 cells, and then the F + recAl strain (XL-1 Blue, Stratagene. San Dieg o, CA), and transforming the resulting transformants with helper M13 phage (VCSM13, Stratagene. San Diego. (CA) at a multiplicity of infection (MOI) of 10. RecA (Clark (1965) Proc. Natl. Aca. Sci. USA 53: 451-459). RecA1 Mutation That Disrupts Sex Is 29kb pAd-L-f1 Stability During Helper Phage Infection Required for Stable high titer of Ad phagemid phage (10^TenMore than Transduction unit) stocks were obtained. Then these ssDNAs with the Ad genome The mutS 201: Tn5 strain (Siegel (1982) Mutat. Res. . 93: 25-33) to promote recombination in vivo. Homologous recombination is It is particularly effective between single-stranded forms of intracellular DNA. After replication, intracellular phagemid Behaves as a normal plasmid, and provides additional positives during subsequent cell growth Subject to mid-plasmid recombination. Shuffling Ad phagemid from cells Finally recovered and purified and used to transfect Hela cells To generate a high titer library.   Phagemid vectors are used for peptide display, cDNA cloning, and Widely used for site-directed mutagenesis (mMead (1988) for review) Biotechnol. 10: 85-102). However, phagemid vectors are large Not used in size (insert) DNA. Conventional phagemid system For cloning of DNA fragments larger than 0 kilobase or larger size ( Not used for the production of ssDNA (10 kb or more). Ad phage of the present invention Do accept inserts as large as 15 and 24 kilobases, and It has been shown to produce ssDNA of the size efficiently. More preferred embodiment In a similar manner, a larger DNA insert as large as 50-100 kb is Inserted into the Ad phagemid; producing full-length ssDNA corresponding to these large inserts. To achieve. The generation of such large ssDNA fragments has progressed throughout the viral genome. (Ie, modification by the recursive recombination method of the present invention) I do. Thus, the present invention clones large DNA inserts (10 KB or more) and To generate ssDNA corresponding to these large inserts in vitro and in vivo First, a unique phagemid system is provided.   The foregoing description of a preferred embodiment of the invention has been presented for purposes of illustration and description. ing. They are intended to be exhaustive of the invention in its precise form, Also, it is not intended to be limiting, and many modifications and variations are possible in light of the above teaching. It is possible. Such modifications and variations that may be apparent to a person skilled in the art are It is intended to be within the scope of the description. All patent documents and publications cited above The works are subject to their purposes for all purposes in the same scope, so that the matter is described separately. The whole is incorporated by reference.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, S D, SZ, UG, ZW), EA (AM, AZ, BY, KG) , KZ, MD, RU, TJ, TM), AL, AM, AT , AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, F I, GB, GE, GH, HU, ID, IL, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, M W, MX, NO, NZ, PL, PT, RO, RU, SD , SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventors Liu, Sea Cow             United States California 95014,             Cupertino, Maplewood Road             10639-A

Claims (1)

[Claims] 1. A method for evolving a nucleic acid segment for use in gene therapy, comprising:         (1) In order to prepare a library of recombinant segments, at least At least a first form and a second form of a segment in which two nucleotides are different from each other. Recombining the two forms;         (2) at least one recombinant segment from the library is Screening for properties useful for gene therapy;         (3) To create a further library of recombinant segments, Recombines at least one recombinant segment with a further form of the segment Wherein the further form is the same or different from the first and second forms. Process         (4) at least one further set from said further library Recombinant segments are screened for the improvement of the property useful for gene therapy. Step;         (5) If necessary, replace (3) and (4) with the additional recombinant segment. Repetition until the agent imparts the desired level of the property useful for gene therapy ;   Wherein the property is:           a) improved titer of a viral vector containing said recombinant segment;           b) Improved infectivity of the viral vector containing said recombinant segment ;           c) increased expression of a gene within the nucleic acid segment;           d) enhanced tissue specificity of the vector containing the recombinant segment;           e) increased genomic yield of the viral vector containing the recombinant segment capacity;           f) Reduction of the recombinant segment or the expression product of the recombinant segment Little immunogenicity;           g) increased stability;           h) transfecting a cell line containing the nucleic acid segment into the cell line Enhanced ability to package purified viral DNA;           i) the nucleus of the rep protein encoded by the nucleic acid segment Reduced toxicity to cell lines expressing the acid segment; and           j) A gene therapy vector for the cell type for which selective removal is desired Increased lethality A method selected from the group consisting of: 2. 2. The method of claim 1, wherein at least one recombination step occurs in vivo. 3. 2. The method of claim 1, wherein at least one recombination step occurs in vitro. . 4. 2. The method of claim 1, wherein said property is tissue specificity and said nucleic acid cell is Fragment comprises a viral nucleic acid segment, and said recombinant segment comprises:         Contacting the virus with cells of a first population and cells of a second population Wherein infectivity by said virus is desired for said first population of cells, Undesired for the second population of cells; and         Isolating a progeny virus from the cells of the first population, the progeny comprising: The virus is a recombinant segment that confers infectivity on cells of the first subpopulation. Enriched process   Screening as a component of the virus. 5. 2. The method of claim 1, wherein the property is a viral genome capacity, The nucleic acid segment comprises a viral nucleic acid segment, and the recombinant segment A virus that propagates the virus in the cell and contains the recombinant segment. By isolating the progeny virus, it is screened as a component of the virus. And the method comprises the steps of: The method further comprising increasing the genome length of the virus comprising. 6. 2. The method of claim 1, wherein the property is the recombinant segment or The expression product is reduced immunogenicity and the recombinant segment is mammalian Introducing the recombination segment into the product, and the recombination remaining after a predetermined period of time. The method, wherein the method is screened by retrieving the segment. 7. 2. The method of claim 1, wherein the property is increased stability, and Subjecting the virus to destabilizing conditions; and Screening as a component of the virus by recovering the virus Wherein the remaining virus is enriched in the recombinant segment conferring the property. Is the way. 8. 2. The method of claim 1, wherein said nucleic acid segment is a viral protein. Wherein the property is a transgenic cell line comprising the nucleic acid segment. Enhanced ability to package infected viral DNA, and The recombinant segment is a cell containing the recombinant segment, Transfection with the strain, and the window produced by various cells. Screening by determining the yield of progeny virus containing the lus vector Where cells giving high yield have increased packaging capacity A method wherein the recombinant segment is enriched. 9. 2. The method of claim 1, wherein the nucleic acid segment is a negative selection gene. A cell which encodes the offspring and whose properties are for the selective elimination of the gene therapy vector. For cell types that are enhanced lethal to the type and for which elimination is not desired Is reduced lethality, and the recombinant segment is         The type of cells desired for selective elimination are transferred to the live recombinant segment. Transfecting with a rally-containing vector and killing the cells Identifying these members of a library of potential recombinant segments; and And         Cells of the type that are not desired to be eliminated are transformed into cells that are not desired to be selectively eliminated. Vectors comprising members of a library of recombinant segments that are lethal to Transfection, and for types of cells not desired to be eliminated Identifying a non-lethal virus   Screening method. 10. The method of claim 9, wherein the negative selection gene is lysine, A polypeptide selected from the group consisting of diphtheria toxin and HSV thymidine kinase The way to code. 11. A method of evolving a cell to package a virus, comprising:         (1) Culture of cells encoding the viral packaging protein Is transformed with a library of DNA fragments, At least some of the library of fragments are homologous in the genome of the cell. Producing a modified cell via recombination with a body segment;         (2) transfecting the modified cells with viral DNA; Determining the yield of progeny virus produced by the cell;         (3) The modified cells giving the highest virus yield were Transforming with a further library, wherein the DNA fragment is further transformed. At least some of the libraries are homologous in the genome of the modified cells. Producing further modified cells via homologous recombination with a body segment;         (4) transfecting the further modified cells with viral DNA; Determining the yield of progeny virus produced by the various cells; and         (5) If necessary, until the further modified cells have acquired the desired function. Repeating steps (3) and (4)   A method comprising: