Heterologous human immunodeficiency virus type 1 lentiviral vectors packaging a simian immunodeficiency virus-derived genome display a specific postentry transduction defect in dendritic cells

Heterologous lentiviral vectors (LVs) represent a way to address safety concerns in the field of gene therapy by decreasing the possibility of genetic recombination between vector and packaging constructs and the generation of replication-competent viruses. Using described LVs based on human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus MAC251 (SIV(MAC251)), we asked whether heterologous virion particles in which trans-acting factors belonged to HIV-1 and cis elements belonged to SIV(MAC251) (HIV-siv) would behave as parental homologous vectors in all cell types. To our surprise, we found that although the heterologous HIV-siv vector was as infectious as its homologous counterpart in most human cells, it was defective in the transduction of dendritic cells (DCs) and, to a lesser extent, macrophages. In DCs, the main postentry defect was observed in the formation of two-long-terminal-repeat circles, despite the fact that full-length proviral DNA was being synthesized and was associated with the nucleus. Taken together, our data suggest that heterologous HIV-siv vectors display a cell-dependent infectivity defect, most probably at a post-nuclear entry migration step. As homologous HIV and SIV vectors do transduce DCs, we believe that these results underscore the importance of a conserved interaction between cis elements and trans-acting viral factors that is lost or suboptimal in heterologous vectors and essential only in the transduction of certain cell types. For gene therapy purposes, these findings indicate that the cellular tropism of LVs can be modulated not only through the use of distinct envelope proteins or tissue-specific promoters but also through the specific combinatorial use of packaging and transfer vector constructs.     

Efficiency of insertion versus replacement vector targeting varies at different chromosomal loci.

We have analyzed the targeting frequencies and recombination products generated with isogenic vectors at the fah and fgr loci in embryonic stem cells. A single vector which could be linearized at different sites to generate either a replacement or an insertion vector was constructed for each locus. A replacement event predominated when the vectors were linearized at the edge of the homologous sequences, while an insertion event predominated when the vectors were linearized within the homologous sequences. However, the ratio of the targeting frequencies exhibited by the different vector configurations differed for the two loci. When the fgr vector was linearized as an insertion vector, the ratio of targeted to random integrations was four- to eightfold greater than when the vector was linearized as a replacement vector. By contrast, the ratio of targeted to random integrations at the fah locus did not vary with the linearization site of the vector. The different relationships between the targeting frequency and the vector configuration at the fgr and fah loci may indicate a DNA sequence or chromatin structure preference for different targeting pathways.     

Inverted terminal repeat sequences are important for intermolecular recombination and circularization of adeno-associated virus genomes

The relatively small package capacity (less than 5 kb) of adeno-associated virus (AAV) vectors has been effectively doubled with the development of dual-vector heterodimerization approaches. However, the efficiency of such dual-vector systems is limited not only by the extent to which intermolecular recombination occurs between two independent vector genomes, but also by the directional bias required for successful transgene reconstitution following concatemerization. In the present study, we sought to evaluate the mechanisms by which inverted terminal repeat (ITR) sequences mediate intermolecular recombination of AAV genomes, with the goal of engineering more efficient vectors for dual-vector trans-splicing approaches. To this end, we generated a novel AAV hybrid-ITR vector characterized by an AAV-2 and an AAV-5 ITR at opposite ends of the viral genome. This hybrid genome was efficiently packaged into either AAV-2 or AAV-5 capsids to generate infectious virions. Hybrid AV2:5 ITR viruses had a significantly lower capacity to form circular intermediates in infected cells than homologous AV2:2 and AV5:5 ITR vectors despite their similar capacity to express an encoded enhanced green fluorescent protein (EGFP) transgene. To examine whether the divergent ITR sequences contained within hybrid AV2:5 ITR vectors could direct intermolecular recombination in a tail-to-head fashion, we generated two hybrid ITR trans-splicing vectors (AV5:2LacZdonor and AV2:5LacZacceptor). Each delivered one exon of a beta-galactosidase minigene flanked by donor or acceptor splice sequences. These hybrid trans-splicing vectors were compared to homologous AV5:5 and AV2:2 trans-splicing vector sets for their ability to reconstitute beta-galactosidase gene expression. Results from this comparison demonstrated that hybrid ITR dual-vector sets had a significantly enhanced trans-splicing efficiency (6- to 10-fold, depending on the capsid serotype) compared to homologous ITR vectors. Molecular studies of viral genome structures suggest that hybrid ITR vectors provide more efficient directional recombination due to an increased abundance of linear-form genomes. These studies provide direct evidence for the importance of ITR sequences in directing intermolecular and intramolecular homologous recombination of AAV genomes. The use of hybrid ITR AAV vector genomes provides new strategies to manipulate viral genome conversion products and to direct intermolecular recombination events required for efficient dual-AAV vector reconstitution of the transgene.     

Illegitimate recombination in a bovine papillomavirus shuttle vector: a high level of site specificity.

Recombination in a bovine papillomavirus shuttle vector carrying direct repeats of Moloney murine leukemia virus LTR sequence was examined. Differently from similar vectors carrying direct repeats of SV40 polyA addition signal or neomycin resistance gene, the vector exhibited no homologous recombination between the repeats. Instead, illegitimate recombination took place. There were two major types of recombination products from the restriction cleavage pattern. The plasmids in independent cellular clones belonging to the same recombination type shared the identical crossover point. Thus, in this plasmid, illegitimate recombination occurred at preferential sites involving exactly the same sequences.     

Characterization of in vivo recombination activities in the mouse embryo

Homologous recombination makes use of sequence homology to repair DNA and to rearrange genetic material. In mammals, these processes have mainly been characterized using cultured cell systems. We have developed an assay that allows us to quantitatively analyze homologous recombination in vivo in the mouse embryo. Transgenic mouse lines were generated by microinjection into a fertilized mouse ovum of a vector containing two homologous LINE-1 (L1) sequences arranged as a direct repeat: these sequences can recombine with each other and with endogenous L1 sequences before, during or after integration of the vector into the genome. Using a plasmid rescue procedure, we determined the composition of the integrated vector array in several transgenic mice and their descendants. Homologous recombination frequencies were found to be strikingly high, involving 70% of integrated vectors in some arrays, with homologous deletions being five times more frequent than gene conversion without crossing-over. Interestingly, non-homologous recombination was found to be much less frequent. We also found that endogenous L1 sequences could be involved in homologous recombination events in the mouse embryo, and that the integrated arrays could be modified from generation to generation by homologous recombination between the integrated L1 sequences.     

Expression and recombination of the EGFP and EYFP genes in lentiviral vectors carrying two heterologous promoters

BACKGROUND: Expressing two genes in the progeny of stem and progenitor cells that are transduced with a unique viral vector is desirable in certain situations. We tested the ability of two lentiviral vectors to transduce human cells of hematopoietic origin and concomitantly express two reporter genes, either EGFP (enhanced green fluorescent protein) and DsRed2, or EGFP and EYFP (enhanced yellow fluorescent protein), from two internal promoters. METHODS: The vectors were generated from the pTRIP deltaU3 EF1alpha EGFP lentiviral vector. Following transduction of hematopoietic and non-hematopoietic cell lines, we performed FACS, PCR and Southern blot analyzes to quantify transduction, integration efficiencies and size of integrated lentiviral vectors, respectively. RESULTS: The detection of DsRed2 fluorescence appeared unexpectedly low in human cells of hematopoietic origin. Alternatively, a modification in the flow cytometry assay allowed us to distinguish between the two overlapping fluorescence signals emitted by EGFP and EYFP, when transduced cells were excited with a 488-nm laser beam. However, the low frequency of double-positive EGFP+ EYFP+ cells, and the existence of single-positive, mostly EGFP- EYFP+, cells, prompted us to search for recombinations in the vector sequence. Southern blotting of DNA obtained from transduced cells indeed demonstrated that recombination had occurred between the two closely related EGFP and EYFP sequences. DISCUSSION: These observations suggest that recombination occurred within the EGFP and EYFP genes, which differ by only four amino acids. We conclude that the insertion of two highly homologous sequences into a lentiviral backbone can favor recombination.     

Replacement-like recombination induced by an integration vector with a murine homology flank at the immunoglobulin heavy-chain locus in mouse and rat hybridoma cells

Vectors for homologous recombination are commonly designed as replacement or integration constructs. We have evaluated integration vectors for the substitution of the immunoglobulin heavy-chain constant region by various human isotypes in mouse and rat hybridomas. It is known that under certain circumstances replacement vectors exhibit a lower target efficiency and can be incorporated by integration events. Conversely, we show here that an integration vector can undergo a replacement event despite having free homologous adjacent DNA ends, which would be expected to initiate integration according to the double-strand break repair model. Moreover, in cases of replacement recombination the 5 crossover is not necessarily located within the homology region, thereby giving rise to a truncated gene product. Whether or not the replacement leads to such deletions is clearly dependent on the isotypes involved in the targeting reaction. The fact that the vector is correctly targeted to the heavy-chain locus, but that the homology region is not always the site of recombination, points to a novel recombination mechanism that may be specific for the immunoglobulin loci and that seems to be predominant even in the presence of the free homologous adjacent ends of an integration vector. Furthermore we demonstrate that homologous recombination at the heavy-chain locus is also possible between sequences from different species. The implications of our findings for the production of chimeric antibodies are discussed.     

Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells.

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.     

Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex.

We describe an efficient cloning system utilizing adenoviral DNA-protein complexes which allows the directional cloning of genes into adenoviral expression vectors in a single step. DNA-protein complexes derived from a recombinant adenovirus (AVC2.null) were isolated by sequential use of CsCl step gradients followed by isopycnic centrifugation in a mixture of CsCl and guanidine HCl. AVC2.null is an adenoviral expression vector containing unique restriction sites between the human CMV-IE promoter and the SV40 intron/polyadenylation site. Transgenes were prepared for cloning into this vector by introduction of compatible restriction sites by PCR. A vector expressing rat granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed using DNA-protein complex as well as by traditional recombination techniques. The efficacy of our adenoviral cloning system utilizing DNA-protein complex was two logs higher than that seen using homologous recombination. All viruses generated by directional ligation of the insert into the vector DNA-protein complexes contained the desired transgene in the correct orientation. This technique greatly simplifies and accelerates the generation of recombinant adenoviral vectors.     

Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction

Salmonella typhimurium bacteriophage P22 transduced plasmids having P22 sequences inserted in the vector pBR322 with high frequency. Analysis of the structure of the transducing particle DNA and the transduced plasmids indicates that this plasmid transduction involves two homologous recombination events. In the donor cell, a single recombination between the phage and the homologous sequences on the plasmid inserted the plasmid into the phage chromosome, which was then packaged by headfuls into P22 particles. The transducing particle DNA contained duplications of the region of homology flanking the integrated plasmid vector sequences and lacked some phage genes. When these defective phage genomes containing the inserted plasmid infected a recipient cell, recombination between the duplicated regions regenerated the plasmid. A useful consequence of this sequence of events was that genetic markers in the region of homology were readily transferred from phage to plasmid. Plasmid transduction required homology between the phage and the plasmid, but did not depend on the presence of any specific P22 sequence in the plasmid. When the infecting P22 carried a DNA sequence homologous to the ampicillin resistance region of pBR322, the vector plasmid having no P22 insert could be transduced. P22-mediated transduction is a useful way to transfer chimeric plasmids, since most S. typhimurium strains are poorly transformed by plasmid DNA.     

Lentivirus vectors using human and simian immunodeficiency virus elements

Lentivirus vectors based on human immunodeficiency virus (HIV) type 1 (HIV-1) constitute a recent development in the field of gene therapy. A key property of HIV-1-derived vectors is their ability to infect nondividing cells. Although high-titer HIV-1-derived vectors have been produced, concerns regarding safety still exist. Safety concerns arise mainly from the possibility of recombination between transfer and packaging vectors, which may give rise to replication-competent viruses with pathogenic potential. We describe a novel lentivirus vector which is based on HIV, simian immunodeficiency virus (SIV), and vesicular stomatitis virus (VSV) and which we refer to as HIV/SIVpack/G. In this system, an HIV-1-derived genome is encapsidated by SIVmac core particles. These core particles are pseudotyped with VSV glycoprotein G. Because the nucleotide homology between HIV-1 and SIVmac is low, the likelihood of recombination between vector elements should be reduced. In addition, the packaging construct (SIVpack) for this lentivirus system was derived from SIVmac1A11, a nonvirulent SIV strain. Thus, the potential for pathogenicity with this vector system is minimal. The transduction ability of HIV/SIVpack/G was demonstrated with immortalized human lymphocytes, human primary macrophages, human bone marrow-derived CD34(+) cells, and primary mouse neurons. To our knowledge, these experiments constitute the first demonstration that the HIV-1-derived genome can be packaged by an SIVmac capsid. We demonstrate that the lentivirus vector described here recapitulates the biological properties of HIV-1-derived vectors, although with increased potential for safety in humans.     

Parameters controlling the rate of gene targeting frequency in the protozoan parasite Leishmania.

In this study we investigated the role of several parameters governing the efficiency of gene targeting mediated by homologous recombination in the protozoan parasite Leishmania. We evaluated the relative targeting frequencies of different replacement vectors designed to target several sequences within the parasite genome. We found that a decrease in the length of homologous sequences <1 kb on one arm of the vector linearly influences the targeting frequency. No homologous recombination was detected, however, when the flanking homologous regions were <180 bp. A requirement for a very high degree of homology between donor and target sequences was found necessary for efficient gene targeting in Leishmania , as targeted recombination was strongly affected by base pair mismatches. Targeting frequency increased proportionally with copy number of the target only when the target was part of a linear amplicon, but remained unchanged when it was present on circles. Different chromosomal locations were found to be targeted with significantly variable levels of efficiency. Finally, different strains of the same species showed differences in gene targeting frequency. Overall, gene targeting mediated by homologous recombination in Leishmania shares similarities to both the yeast and the mammalian recombination systems.     

New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors.

We prepared retrovirus packaging cell lines containing gag-pol genes from spleen necrosis virus (expressed from a cytomegalovirus promoter and the simian virus 40 (SV40) polyadenylation sequences) and, on a separate vector, either the env gene from spleen necrosis virus (expressed from the Rous sarcoma virus promoter and the SV40 polyadenylation sequences) or the env gene from amphotropic murine leukemia virus (expressed from a cytomegalovirus promoter and the SV40 polyadenylation sequences). The nucleotide sequences in these packaging cell lines have almost no homology to the retrovirus vectors we used. Retrovirus vectors were produced from these new helper cell lines without any genetic interactions between the vectors and sequences in the helper cells and without transfer of the packaging sequences.     

Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism.

To expand the utility of recombinant adenovirus vectors for gene therapy applications, methods to alter native viral tropism to achieve cell-specific transduction would be beneficial. To this end, we are pursuing genetic methods to alter the cell recognition domain of the adenovirus fiber. To incorporate these modified fibers into mature virions, we have developed a method based on homologous DNA recombination between two plasmids. A fiber-deleted, propagation-defective rescue plasmid has been designed for recombination with a shuttle plasmid encoding a variant fiber gene. Recombination between the two plasmids results in the derivation of recombinant viruses containing the variant fiber gene. To establish the utility of this method, we constructed a recombinant adenovirus containing a fiber gene with a silent mutation. In addition, we generated an adenovirus vector containing chimeric fibers composed of the tail and shaft domains of adenovirus serotype 5 and the knob domain of serotype 3. This modification was shown to alter the receptor recognition profile of the virus containing the fiber chimera. Thus, this two-plasmid system allows for the generation of adenovirus vectors containing variant fibers. This method provides a rapid and facile means of generating fiber-modified recombinant adenoviruses. In addition, it should be possible to use this system in the development of adenovirus vectors with modified tropism to allow cell-specific targeting.     

Integration of heterologous plasmid DNA into multiple sites on the genome of Campylobacter coli following natural transformation.

The efficiency of homologous recombination in Campylobacter coli following the introduction of DNA by natural transformation was determined by using a series of nonreplicating integrative vectors containing DNA fragments derived from the C. coli catalase gene. Homologous recombination occurred with as little as 286 homologous bp present and was not detected when 270 bases of homology was provided. Instead, when plasmids with little or no homology to the chromosome were introduced by natural transformation, the vector DNA became chromosomally integrated at random sites scattered throughout the C. coli genome. Southern analysis and nucleotide sequencing revealed that recombination had occurred between nonhomologous sequences and can therefore be described as illegitimate. There were at least five different recombination sites on plasmid pSP105. The ability of C. coli to acquire heterologous plasmids by natural transformation, and maintain them by chromosomal integration following illegitimate recombination, has fascinating implications for the genomic diversity and evolution of this species.     

Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells

Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5′- and 3′-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis.     

Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning

The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes. The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest. The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) containing the gene of interest. To further enhance and refine the TAR cloning technology, we determined the minimal size of a specific hook required for gene isolation utilizing the Tg.AC mouse transgene as a targeted region. For this purpose a set of vectors containing a B1 repeat hook and a Tg.AC-specific hook of variable sizes (from 20 to 800 bp) was constructed and checked for efficiency of transgene isolation by a radial TAR cloning. When vectors with a specific hook that was ≥60 bp were utilized, ~2% of transformants contained circular YACs with the Tg.AC transgene sequences. Efficiency of cloning dramatically decreased when the TAR vector contained a hook of 40 bp or less. Thus, the minimal length of a unique sequence required for gene isolation by TAR is ~60 bp. No transgene-positive YAC clones were detected when an ARS element was incorporated into a vector, demonstrating that the absence of a yeast origin of replication in a vector is a prerequisite for efficient gene isolation by TAR cloning.