Vector Proviruses Research Articles

Short Communication: Activation of Latent HIV Type 1 Gene Expression by Suberoylanilide Hydroxamic Acid (SAHA), an HDAC Inhibitor Approved for Use to Treat Cutaneous T Cell Lymphoma

The ability of HIV to establish a latent infection causes life-long virus persistence, even after long-term highly active antiretroviral therapy (HAART). The role that latency is playing in preventing clearance of the virus infection has become evident in recent years. Patients who have been successfully treated with ART, having undetectable levels of viral RNA (below 50 copies/ml) in the plasma for years, experienced rapid virus rebound on withdrawal of therapy. Activation of latent proviruses from the infected cells in combination with ART is a therapeutic strategy that may lead to the complete elimination of HIV infection. We report here that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor that has been approved for the treatment of cutaneous T cell lymphoma (CTCL), can activate an HIV-1 vector provirus in a cell model system. Treatment of cells harboring a latent, HIV-1-derived provirus caused activation of both early and late viral gene expression, acetylation of nucleosome on the 5' long terminal repeat (LTR), and remodeling of the chromatin at the 5' LTR. Several compounds, including valproic acid, have been tested for their ability to activate latent HIV-1, but have met with disappointing results. SAHA, a relatively nontoxic, FDA-approved compound, should be considered for developing a strategy to eliminate HIV from patients.

Genomic and Functional Assays Demonstrate Reduced Gammaretroviral Vector Genotoxicity Associated With Use of the cHS4 Chromatin Insulator

Interest in the use of recombinant retroviral vectors for clinical gene therapy has been tempered by evidence of vector-mediated genotoxicity involving the activation of cellular oncogenes flanking sites of vector integration. We report here that the rate of gammaretroviral vector genotoxicity can be significantly reduced by addition of the cHS4 chromatin insulator, based on two complementary approaches for assessing vector-mediated genotoxicity. One approach involves the direct, genomewide assessment of cellular gene dysregulation using panels of transduced cell clones and genomic microarrays, whereas the other involves the functional assessment of malignant transformation using a factor-dependent cell line. Both assays are robust and quantitative, and indicate the cHS4 chromatin insulator can reduce vector-mediated genotoxicity approximately sixfold (ranged three to eight fold). These approaches also provide a means for assessing various aspects of vector-mediated genotoxicity, including the overall rate of cellular gene dysregulation, the potential influence of vector provirus over large genomic distances, and the involvement of oncogenic pathways in vector-mediated malignant transformation.

A Rapid and Quantitative Assay for Measuring Neighboring Gene Activation by Vector Proviruses

A simple, quantitative assay for measuring the oncogenic potential of integrating vectors is needed in order to improve vector design and safety. In this study, we have developed a transient plasmid-based assay to measure the activation of a reporter gene by an adjacent vector provirus. Plasmid pACT contains a luciferase cassette driven by a minimal, enhancerless promoter, into which vector proviruses are inserted upstream for evaluation by luciferase assays and northern blots. In a comparison of analogous vectors based on murine leukemia virus (MLV), human immunodeficiency virus (HIV), and foamy virus (FV), we observed significant enhancer activity and read-through transcription from MLV proviruses, and significant read-through transcription from HIV proviruses. HIV and FV proviruses containing an internal MLV long-terminal repeat (LTR) promoter also had significant enhancer activity, which was not observed with an internal promoter from the murine phosphoglycerate kinase-1 gene, PGK. These results demonstrate that neighboring gene activation can be limited by using internal promoter(s) lacking enhancer activity, especially when present in an FV vector backbone that prevents read-through transcription. Although the pACT assay does not measure oncogenesis directly, it should be useful for screening vectors before more time-consuming and costly animal studies are undertaken.

Distinctive Integration Profile of Avian Sarcoma Leukosis Virus Vectors in Rhesus Long-Term Repopulating Cells.

Insertional mutagenesis continues to be a major concern in hematopoietic stem cell gene therapy. Non-conventional gene transfer vectors with more favorable integration features in comparison to conventional retrovirus and lentivirus vectors are being pursued. The avian sarcoma leukosis virus (ASLV) has been reported to have an unbiased integration pattern in cell lines, but this vector system has not previously been investigated for transduction of repopulating hematopoietic cells. We investigated the efficiency and integration profile of this vector in the clinically-relevant rhesus macaque autologous transplantation model. Using an ASLV derived RCAS (Replication Competent, ALV LTR with A Splice acceptor) vector, we could obtain transduction efficiencies of up to 33% ex vivo in rhesus CD34+ cells. We have been able to transplant two rhesus macaques with ASLV-transduced autologous CD34+ cells and achieve long-term gene marking levels of 1–3% as far as 18 months post-transplant. Using an optimized linear-amplification mediated PCR (LAM-PCR), we have been able to identify so far close to 300 unique insertion sites in the two animals. Here we reported for the first time a systematic analysis of 239 ASLV insertion sites identified in rhesus long-term repopulating cells. Out of 239 unique insertions identified in 4–18 months post-transplant granulocytes and lymphocytes, 99 (41.1%) have landed within RefSeq gene coding regions. 14 (5.8%) have landed within 5kb upstream of RefSeq genes, indicating no preference of inserting into transcription start site. 10 (4.2%) have landed within 5kb downstream of RefSeq genes, which is comparable to random insertions. No insertion into the Mds1-Evi1 locus has been identified to date, at any time point, which is in striking contrast to significant overrepresentation of this insertion site for MLV vectors in the same transplantation model. No preference into CpG islands was found. We further employed a rapid and quantitative assay for measuring neighboring gene activation by vector provirus LTR enhancers using a luciferase assay. LTR from vectors are cloned into the pACT5 plasmid, upstream of a minimal promoter-IRES-luciferease cassette, allowing measurement of neighboring gene activation by a simple luciferase assay. RCAS vectors produced no detectable luciferase activation, both in the forward and reverse orientations, suggesting lack of enhancer activity in the LTRs in mammalian cells, in contrast to significant enhancer activity and read-through transcription from MLV vectors measured in the same assay. Our findings indicate that ASLV derived RCAS vectors have a potentially safer integration pattern in comparison to commonly used MLV and HIV derived vectors. Furthermore, ASLV LTRs do not have significant promoter and enhancer activity in mammalian cells, which provide a second safety feature of the system. Therefore, these vectors should be further explored for hematopoietic stem cell gene transfer purposes.

AAV Vectors, Insertional Mutagenesis, and Cancer

By now we have all heard a lot about retroviral vectors and cancer, but other types of vectors have typically been left out of these discussions. However, results presented at the recent American Society of Gene Therapy meeting and just published in Science1 show that this is a subject that goes beyond retroviruses, hematopoietic cells, and gamma C transgenes. In this study, mice developed hepatocellular carcinomas (HCCs) that contained adeno-associated virus (AAV) vector proviruses at a specific chromosomal locus, raising important questions about the safety of in vivo gene therapy.

Development of Leukemia after HOXB4 Gene Transfer in the Canine Model.

HOXB4 is considered to be the only HOX gene that promotes self-renewal of hematopoietic stem cells without causing leukemia in mouse models. We investigated whether HOXB4 overexpression has similar effects in a clinically relevant canine model. A competitive repopulation assay was performed in three dogs in which CD34+ cells were transduced with MSCV-based gammaretroviral vectors expressing HOXB4GFP or control YFP. We observed up to 4-fold higher marking levels in granulocytes for the HOXB4GFP arm relative to the control 1 month after transplantation. The marking levels eventually decreased in all three animals and two dogs (G374, G450) have now been followed for more than 18 months. In G374, the marking levels for both arms stabilized at ~2% after 2 months post-transplantation. Between 14 and 20 months post-transplantation, the HOXB4GFP marking steadily increased to >95%, while YFP marking decreased to 0.1%. G374 was euthanized 21 months after transplantation due to declining health. Flow cytometry analysis showed that ~50% of BM cells expressed the monocyte marker CD14 and ~8% expressed the granulocyte marker DM5, all of which also expressed HOXB4GFP. CD3 and CD21 were expressed in 2% and 1% of cells, respectively, but these cells did not express HOXB4GFP. Bone marrow necropsy demonstrated significantly increased numbers of blast cells, consistent with a myelomonocytic leukemia. Southern blot analyses of G374 BM and PB samples identified 2 bands with the same intensity, suggesting a single dominant clone with 2 integration sites. LAM-PCR analysis identified two vector proviruses integrated ~100 kb upstream of c-myb, and into intron 3 of PRDM16. Western blot analysis confirmed expression of HOXB4 in cultured G374 BM cells but the levels of c-myb in these cells were not different from control HOXB4-transduced BM cells as determined by RT-PCR. The expression of PRDM16 exons 1–3 was not detected in cells from dog G374 or in control cells, however, PRDM16 exon 4 was expressed in G374 cells but not in control cells. RT-PCR using primers located in the MSCV LTR and in PRDM16 exon 4 identified a unique band and sequencing of this product showed that the 5′ LTR was spliced with PRDM16 exon 4 creating a short PRDM16 isoform which has been observed in human leukemias. These data suggest that HOXB4 overexpression in collaboration with integration-induced activation of PRDM16 led to the leukemia. Southern blot and SYBR green Q-PCR showed that the leukemic clone contributed to ~20% hematopoiesis in BM 6 months after transplantation, and gradually decreased to ~2% before final expansion of the clone, suggesting accumulation of other mutation(s) were required for overt leukemia. Karyotype analysis of BM cells has not shown any major abnormalities but we are currently performing analyses to search for minor abnormalities such as gene duplications and deletions. Recently, HOXB4GFP marking in dog G450 PB and BM has increased to 20% and 80%, respectively, while YFP marking has decreased to ~1%. Southern blot analysis has identified a single dominant band and a BM biopsy showed substantially increased blast cells. Of note, we have not observed leukemia in >30 dogs followed long term that received transduced cells without HOXB4. In summary, HOXB4 overexpression together with insertional mutagenesis by virus integration has induced leukemia in the canine model, demonstrating the utility of this model to study the safety of gene therapy.

Coordinate enhancement of transgene transcription and translation in a lentiviral vector

BackgroundCoordinate enhancement of transgene transcription and translation would be a potent approach to significantly improve protein output in a broad array of viral vectors and nonviral expression systems. Many vector transgenes are complementary DNA (cDNA). The lack of splicing can significantly reduce the efficiency of their translation. Some retroviruses contain a 5' terminal post-transcriptional control element (PCE) that facilitates translation of unspliced mRNA. Here we evaluated the potential for spleen necrosis virus PCE to stimulate protein production from HIV-1 based lentiviral vector by: 1) improving translation of the internal transgene transcript; and 2) functionally synergizing with a transcriptional enhancer to achieve coordinate increases in RNA synthesis and translation.ResultsDerivatives of HIV-1 SIN self-inactivating lentiviral vector were created that contain PCE and cytomegalovirus immediate early enhancer (CMV IE). Results from transfected cells and four different transduced cell types indicate that: 1) PCE enhanced transgene protein synthesis; 2) transcription from the internal promoter is enhanced by CMV IE; 3) PCE and CMV IE functioned synergistically to significantly increase transgene protein yield; 4) the magnitude of translation enhancement by PCE was similar in transfected and transduced cells; 5) differences were observed in steady state level of PCE vector RNA in transfected and transduced cells; 6) the lower steady state was not attributable to reduced RNA stability, but to lower cytoplasmic accumulation in transduced cells.ConclusionPCE is a useful tool to improve post-transcriptional expression of lentiviral vector transgene. Coordinate enhancement of transcription and translation is conferred by the combination of PCE with CMV IE transcriptional enhancer and increased protein yield up to 11 to 17-fold in transfected cells. The incorporation of the vector provirus into chromatin correlated with reduced cytoplasmic accumulation of PCE transgene RNA. We speculate that epigenetic modulation of promoter activity altered cotranscriptional recruitment of RNA processing factors and reduced the availability of fully processed transcript or the efficiency of export from the nucleus. Our results provide an example of the dynamic interplay between the transcription and post-transcription steps of gene expression and document that introduction of heterologous gene expression signals can yield disparate effects in transfected versus transduced cells.

Foamy virus vector integration sites in normal human cells

Foamy viruses (FVs) or spumaviruses are retroviruses that have been developed as vectors, but their integration patterns have not been described. We have performed a large-scale analysis of FV integration sites in unselected human fibroblasts (n = 1,008) and human CD34(+) hematopoietic cells (n = 1,821) by using a bacterial shuttle vector and a comparable analysis of lentiviral vector integration sites in CD34(+) cells (n = 1,331). FV vectors had a distinct integration profile relative to other types of retroviruses. They did not integrate preferentially within genes, despite a modest preference for integration near transcription start sites and a significant preference for CpG islands. The genomewide distribution of FV vector proviruses was nonrandom, with both clusters and gaps. Transcriptional profiling showed that gene expression had little influence on integration site selection. Our findings suggest that FV vectors may have desirable integration properties for gene therapy applications.

1057. The cHS4 Chromatin Insulator Blocks Epigenetic Interactions between Integrated Vectors and the Surrounding Genome

Gene transfer vectors that integrate at ectopic locations throughout the target cell genome may experience unfavorable interactions with flanking chromosomal elements, including epigenetic silencing of transferred vectors by mechanisms such as encroachment of heterochromatin, or inadvertent activation of cellular genes, such as oncogenes, through the action of enhancers within the vectors. We have been investigating the cHS4 chromatin insulator for the ability to block both types of vector-genome interactions. Chromatin insulators such as cHS4 are DNA elements that act in cis to form functional boundaries between differentially expressed chromosomal loci, blocking both positive effects of enhancers and repressive effects of heterochromatin. Building on studies reported last year, we have found that the ability of the cHS4 element to increase the frequencyof expression from a gammaretrovirus reporter vector 4-fold (P=0.01) following bone marrow transduction and transplantation in mice is associated with two significant epigenetic changes in the vector provirus: a 3.3-fold increase in the overall level of acetylated histones (from 3.8|[plusmn]|2.4 fold to 14.8|[plusmn]|4.5 fold over a GAPDH control, P=0.005); and a 40% reduction in the frequency of methylated CpG dinucleotides present throughout the provirus (from 23|[plusmn]|15% to 14|[plusmn]|13%, P=0.01), with the most significant reduction occurring within the 5|[prime]|LTR promoter and packaging signal (from 9|[plusmn]|7% to 2|[plusmn]|2%, P=0.006). These results demonstrate that the cHS4 element blocks two key molecular mechanisms of epigenetic vector silencing: histone deacetylation and DNA methylation. In a separate set of studies, we assessed the ability of the cHS4 element to reduce the impact of vectors on the expression of cellular genes flanking integration sites. For this purpose, we transduced the human cell line HT1080 with a gammaretrovirus reporter vector, generated a large panel of unique clones in the absence of selection, and analyzed these clones for the presence of disregulated genes using high-density expression arrays and two separate informatics packages. We identified a total of 25 disregulated genes out of 45 cell clones containing 163 provirus for the uninsulated vector, versus 10 disregulated genes out of 42 cell clones containing 145 provirus for the insulated vector. The majority of disregulated genes (65%) exhibited an increased in expression and all disregulated genes were confirmed independently by RT/PCR. This represents a greater than 50% reduction in the frequency of disregulated cellular genes, from 14.7 per 100 provirus to 6.9 per 100 provirus (P=0.002), as a result of flanking the vector with the cHS4 insulator. To date we have directly correlated 9 disregulated genes with specific, mapped vector integration sites. Studies are underway to map the remaining integration sites with the goal of identifying the mechanisms of vector-mediated gene disregulation. Taken together, these studies demonstrate that the cHS4 chromatin insulator can reduce both the epigenetic mechanisms that mediate the silencing effects of flanking chromatin on expression of integrated vectors, and the frequency with which vector integration results in the disregulation of flanking cellular genes.

A Rapid Method for Comparing the Oncogenic Potential of Retroviral Vectors.

Retroviral gene transfer has successfully cured patients with X-linked severe combined immunodeficiency (X-SCID). Unfortunately, three of the treated patients developed leukemia at least in part due to insertional activation of a neighboring gene. Further advances of this exciting therapeutic modality will require safer viral vectors. Genotoxicity experiments in animals, although an important preclinical study, are not practical for screening large numbers of vectors. We have developed a rapid plasmid-based assay to compare downstream gene activation by retroviral vectors. We constructed a plasmid, pACT, that contains a minimal CMV promoter, an internal ribosome entry site (IRES) and a luciferase reported gene followed by a polyadenylation site. Four different proviral constructs containing identical internal PGK-EGFP expression cassettes were inserted upstream of the minimal promoter and nucleofected (Amaxa Biosystems) into K562 cells. The relative luciferase activation of each provirus was determined and compared to cells nucleofected with the basic pACT plasmid. The relative luciferase activation by a plasmid containing a Moloney murine leukemia virus (MLV) vector provirus with full length long terminal repeats (LTRs) was 144, 1230, and 4525 at 1, 2 and 4 days post nucleofection, respectively. Changing the orientation of the proviral DNA did not significantly change the results, consistent with an enhancer effect from the proviral LTRs. In comparison, the relative luciferase activation by a plasmid containing a self inactivating (SIN) MLV vector provirus with LTR deletions was only 59, 90 and 170, respectively, and was reduced by greater than 90% with the reverse orientation consistent with residual activation by read-through transcription. For plasmids containing the HIV SIN vector provirus, the relative luciferase activation was 48, 45 and 52, respectively at 1, 2 and 4 days post nucleofection, and decreased by >90% with the inverse orientation. The relative luciferase activation for plasmids containing the Foamy Virus (FV) vector provirus, also a “SIN” vector, was the least, only 3, 2 and 6, respectively, at 1, 2 and 4 days post nucleofection. Therefore, all three SIN vectors (MLV, HIV and FV) produced significantly less downstream gene activation than a conventional MLV vector, with the FV vectors being the lowest. Additional studies comparing various internal promoters and vectors will also be presented. These results demonstrate the usefulness of the pACT assay system to identify safer retroviral vectors.

80. Insulation of Oncoretrovirus Vectors with the cHS4 Chromatin Insulator Is Associated with an Open Chromatin Histone Code

We and others have reported that the cHS4 chromatin insulator can improve the expression of integrating gene transfer vectors by reducing the impact of silencing chromosomal position effects. Independent studies indicate that the cHS4 element prevents the encroachment of silencing heterochromatin by recruiting high levels of histone acetyl transferases, which in turn acetylate key histone moieties and enforce an open chromatin structure. This is true even in lineages where the neighboring beta-globin locus is transcriptionaly inactive, suggesting that the chromatin opening activity of the cHS4 element is independent of transcriptional activity in adjoining regions. However, previous studies in mouse bone marrow transduction and transplantation models demonstrated that a certain fraction of vector provirus, although flanked with the cHS4 element, nevertheless fail to express the vector transgene. It is unclear whether this lack of expression is associated with a failure of the cHS4 element to maintain an open chromatin state, or a failure of the vector transgene to take advantage of this open chromatin state. In order to determine whether the cHS4 element increases the frequency of vector expression by increasing the level of histone acetylation, and whether vector silencing is associated with a failure of this activity, we analyzed vector provirus in mice transplanted with uninsulated and insulated reporter vectors by chromatin immunoprecipitation (ChIP). Consistent with our previous studies, we found that 4 mice transplanted with the uninsulated vector expressed the LTR-GFP cassette only 9|[plusmn]|11% of the time, compared to 30|[plusmn]|17% for 5 mice transplanted with the same vector flanked with the cHS4 element. Cross-linked chromatin from steady-state spleen cells was precipitated with antibodies to acetylated histone 3 (H3K9/K14), and the ratio of vector provirus sequences in the precipitated and unprecipitated fractions was determined by quantitative PCR and compared to that of an internal control (GAPDH). We found that chromatin associated with uninsulated provirus was acetylated at only 3.8|[plusmn]|2.4 fold of control, while chromatin associated with insulated provirus was acetylated at 12.3|[plusmn]|6.6 fold of control (p=0.04). More detailed mapping indicated a notable peak of acetylated histone associated with the cHS4 elements in the vector LTRs (averaging 20.4|[plusmn]|10.8 fold of control versus 7.1|[plusmn]|6.3 fold for more internal sequences, p=0.001), while no such bias was observed for the uninsulated vector. In one recipient the insulated vector was only expressed in 3.7% of provirus-containing cells. We found that chromatin associated with provirus in this mouse was only acetylated at 2.9 fold of control, or 5-fold lower than in the other mice of this group (p<<0.001). Taken together, these studies suggest that the cHS4 chromatin insulator increases the frequency of provirus expression by maintaining an open chromatin structure via histone acetylation, that this activity appears to peak near the cHS4 elements, and that the failure of some insulated provirus to be expressed is associated with a failure of this activity. Future studies will be needed to determine whether alternative insulators will provide a more robust level of protection.

Adeno-associated virus vectors integrate at chromosome breakage sites.

Adeno-associated virus (AAV) vectors transduce cells by multiple pathways, including integration at nonhomologous chromosomal locations by an unknown mechanism. We reasoned that spontaneous chromosome breaks may facilitate vector integration and investigated this in cells containing a specific chromosomal double-strand break created by the endonuclease I-SceI or multiple breaks created by treatment with etoposide or gamma-irradiation. Vector proviruses were found at I-SceI cleavage sites, and sequencing of vector-chromosome junctions detected microhomologies, deletions and insertions that were similar when integration occurred spontaneously at random locations or at induced double-strand breaks. Infection with AAV vectors did not increase mutation rates in normal human cells. Our results establish a mechanism for integration and suggest that AAV vectors can integrate at existing chromosome breaks rather than causing breaks themselves, which has implications for their clinical use.

749. Adeno-Associated Virus Vectors Integrate at Chromosome Breakage Sites

Adeno-associated virus (AAV) vectors transduce cells by multiple pathways, including integration at non-homologous chromosomal locations by an unknown mechanism that does not require viral proteins. We reasoned that spontaneous chromosome breaks may facilitate vector integration and investigated this by transducing cells containing a specific chromosomal double strand break created by the endonuclease I-SceI. Vector proviruses were frequently found at I-SceI cleavage sites, and sequencing of vector:chromosome junctions revealed micro-homologies, deletions, and insertions that in some cases could be explained by template-directed DNA synthesis prior to ligation. Similar junctions were observed when integration occurred at random locations, and infection with AAV vectors did not increase mutation rates in normal human cells. Our results establish a mechanism for integration, and suggest that AAV vectors integrate at existing chromosome breaks rather than causing breaks, a finding that has unique implications regarding their clinical use.

Chromosomal effects of adeno-associated virus vector integration.

Adeno-associated virus (AAV) vectors are currently being used in several clinical gene-therapy trials (see the NIH OBA Human Gene Transfer Clinical Trials Database); however, little is known about the chromosomal effects of vector integration. Here we report that integrated vector proviruses are associated with chromosomal deletions and other rearrangements and are frequently located on chromosome 19 (although not at the wildtype AAV integration site).

RNase H Activity Is Required for High-Frequency Repeat Deletion during Moloney Murine Leukemia Virus Replication

It has been postulated that retroviral recombination, like strong stop template switching, requires the RNase H activity of reverse transcriptase. To address this hypothesis, Moloney murine leukemia virus-based vectors, which were designed to test the recombination-related property of direct repeat deletion, were encapsidated in virions engineered to contain phenotypic mixtures of wild-type and RNase H catalytic site point mutant reverse transcriptase. Integrated provirus titers per milliliter were determined for these phenotypically mixed virions, and vector proviruses were screened to determine what percentage contained repeat deletions. The results revealed a steady decline in direct repeat deletion frequency that correlated with decreases in functional RNase H, with greater than fourfold decreases in repeat deletion frequency observed when 95% of virion reverse transcriptase was RNase H defective. Parallel experiments were performed to address effects of molar excesses of RNase H relative to functional DNA polymerase. These experiments demonstrated that increasing the stoichiometry of RNase H relative to the amount of functional DNA polymerase had minimal effects on direct repeat deletion frequency. DNA synthesis was error prone when directed principally by RNase H mutant reverse transcriptase, suggesting a role for RNase H catalytic integrity in the fidelity of intracellular reverse transcription.

Helper-free foamy virus vectors.

Retroviral vectors based on human foamy virus (HFV) have been developed and show promise as gene therapy vehicles. Here we describe a method for the production of HFV vector stocks free of detectable helper virus. The helper and vector plasmid constructs used both lack the HFV bel genes, so recombination between these constructs cannot create a wild-type virus. A fusion promoter that combines portions of the cytomegalovirus (CMV) immediate-early and HFV long terminal repeat (LTR) promoters was used to drive expression of both the helper and vector constructs. The CMV-LTR fusion promoter allows for HFV vector production in the absence of the Bel-1 trans-activator protein, which would otherwise be necessary for efficient transcription from the HFV LTR. Vector stocks containing either neomycin phosphotransferase or alkaline phosphatase reporter genes were produced by transient transfection at titers greater than 10(5) transducing units/ml. G418-resistant BHK-21 cells obtained by transduction with neo vectors contained randomly integrated HFV vector proviruses without detectable deletions or rearrangements. The vector stocks generated were free of replication-competent retrovirus (RCR), as determined by assays for LTR trans-activation and a marker rescue assay developed here for the detection of Bel-independent RCR.

Efficient gene transfer in primitive CD34+/CD38lo human bone marrow cells reselected after long-term exposure to GALV-pseudotyped retroviral vector.

Successful retroviral gene transfer into human hematopoietic stem cells was demonstrated in preliminary clinical trials at low efficiency. We have shown previously that gene transfer into committed hematopoietic progenitor cells is more efficient using a gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector instead of an amphotropic retroviral vector. Here, we have conducted a systematic study of human hematopoietic progenitor cells after extended transduction with a GALV-pseudotyped retroviral vector. CD34+/CD38lo Cells were transduced for 5 days and reselected according to phenotype after culture and analyzed for cell cycle status, long-term culture-initiating cell (LTC-IC) activity, and gene transfer. Reselection of rare, very primitive progenitor cells was successful. Equal to fresh CD34+/CD38lo cells, >90% of reselected CD34+/CD38lo cells were in G0/G1. CD34+/CD38lo reselection enriched for LTC-IC (10-fold), as compared to freshly isolated CD34+/CD38lo cells with excellent specificity (82.7% of total LTC-IC were recovered in the reselected CD34+/CD38lo population) and recovery (62% of initial LTC-IC number in CD34+/CD38lo cells were recovered in the reselected fraction after transduction). Gene transfer into primitive progenitor cells was efficient with 50.5% G418-resistant LTC-IC colonies and more than 40 copies of vector provirus detectable per 100 nuclei of CD34+/CD38lo cells. To our knowledge, this is the first systematic analysis of phenotype, function, and cell cycle demonstrating retroviral gene transfer into rare, very primitive human hematopoietic progenitor cells. The chosen strategy should be of considerable value for analyzing and improving gene therapy of the hematopoietic system.

Adeno-associated virus vector integration junctions.

Vectors derived from adeno-associated virus (AAV) have the potential to stably transduce mammalian cells by integrating into host chromosomes. Despite active research on the use of AAV vectors for gene therapy, the structure of integrated vector proviruses has not previously been analyzed at the DNA sequence level. Studies on the integration of wild-type AAV have identified a common site-specific integration locus on human chromosome 19; however, most AAV vectors do not appear to integrate at this locus. To improve our understanding of AAV vector integration, we analyzed the DNA sequences of several integrated vector proviruses. HeLa cells were transduced with an AAV shuttle vector, and integrated proviruses containing flanking human DNA were recovered as bacterial plasmids for further analysis. We found that AAV vectors integrated as single-copy proviruses at random chromosomal locations and that the flanking HeLa DNA at integration sites was not homologous to AAV or the site-specific integration locus of wild-type AAV. Recombination junctions were scattered throughout the vector terminal repeats with no apparent site specificity. None of the integrated vectors were fully intact. Vector proviruses with nearly intact terminal repeats were excised and amplified after infection with wild-type AAV and adenovirus. Our results suggest that AAV vectors integrate by nonhomologous recombination after partial degradation of entering vector genomes. These findings have important implications for the mechanism of AAV vector integration and the use of these vectors in human gene therapy.

Lack of correlation between basal expression levels and susceptibility to transcriptional shutdown among single-gene murine leukemia virus vector proviruses

Integrated retroviruses or retroviral vectors may be transcriptionally inactive although their promoter-enhancer regions are able to direct transcription in the host cell. We have used single-gene retroviral vectors with a long terminal repeat-directed neo marker gene to determine if the level of transcription relates to the susceptibility of a provirus to inactivation. We used two isogenic vectors, carrying long terminal repeats with a strong and a weak transcriptional enhancer derived from SL3-3 and Akv murine leukemia viruses, respectively. Nonselected cell clones of the murine lymphoid cell line L691 with single integrated vector proviruses exhibiting a 20-fold range of initial expression levels were studied. The basal expression level of a given cell clone with a single provirus did not show any pattern of correlation with the long-term stability of expression, as monitored for periods up to 150 days. Our results thus indicate that the inactivation mechanism operates independently of the initial transcriptional activity of the provirus.

Peripheral Blood Lymphocytes as Target Cells of Retroviral Vector-Mediated Gene Transfer

AbstractPeripheral blood lymphocytes (PBLs) are key target cells for gene therapy of a number of inherited and acquired blood disorders. We have systematically compared four retroviral vectors, designed according to different strategies, for their efficiency in transfer and expression in human PBLs of the same reporter gene. The receptor gene used in the study codes for the human low-affinity nerve growth factor receptor (LNGFR), and is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transduced gene expression by immunofluorescence, with single cell resolution. Peripheral blood mononuclear cells (PBMCs), as well as human hematopoietic cell lines of myeloid and lymphoid origin, were transduced with the four vectors and analyzed for efficiency of gene transfer, integration and stability of vector proviruses, and LNGFR expression at both RNA and protein level. Fluorescence-activated cell sorter analysis of coexpression of LNGFR and lineage-specific cell surface markers was performed in transduced cell lines, PBLs, and T-cell clones to study gene expression on specific cell subpopulations. Although crucial differences were observed among different constructs, all retroviral vectors could transduce, under appropriate infection conditions, T-cell populations representative of the normal immune repertoire. Gene transfer and expression could be demonstrated also in circulating progenitors of mature T cells. Expression of the transduced gene was heterogeneous among cell populations infected with the different vectors, with optimal results obtained by two of the four constructs. Finally, we have devised a simple protocol based on vector-mediated gene transfer and positive immunoselection of the transduced cells that produces virtually 100% gene-modified cells. This may represent a crucial improvement in the way of designing efficacious protocols involving the use of gene-modified T lymphocytes in clinical studies.