Dusty Miller

Vectors based on adeno-associated virus (AAV) are under investigation for use in gene therapy applications. Critical aspects of AAV vector biology remain undefined, in particular the intracellular events and activities mediating transduction and determining host cell permissiveness for transduction. Using cultured primary human fibroblasts, we previously showed that AAV vectors preferentially, but not exclusively, transduce cells in the S phase of the cell cycle, and that transduction can be markedly enhanced by pretreatment of target cells with physical and chemical agents that perturb DNA metabolism. In this study, we tested whether similar improvements in AAV vector performance might be achievable in vivo. The adult rat brain and overlying scalp muscle were selected for vector inoculation because of the presence of well-defined populations of dividing, quiescent, and post-mitotic cells, and gamma irradiation was chosen as a reproducible means of inducing DNA repair in these cells. We find that gamma irradiation markedly enhances the transduction of dividing cell populations in the pia-arachnoid and choroid epithelium within the central nervous system, and of mature nondividing muscle cells in the scalp, whereas gamma irradiation did not increase the basal transduction level of post-mitotic neurons in the hippocampus. These data confirm that replicative cellular DNA synthesis is not required for transduction by AAV vectors and show that the mitotic state of target cells is not necessarily predictive of responsiveness to transduction-enhancing treatments. Most importantly, these data demonstrate that target cells can be manipulated in vivo to render them more permissive for AAV vector transduction.

ABSTRACT Gene therapy of the lung requires the introduction and expression of a therapeutic gene in airway cells. Although retroviral vectors may be useful in this context, the ability of retroviruses to infect specific cell types in the airway is not known. In this study, we examined the ability of amphotropic recombinant retroviral vectors to transduce primary cultures of rabbit airway epithelial cell populations purified for basal or secretory cells. Transduction efficiencies in basal and secretory cell populations were found to be similar; about 27% after a single exposure to vector, and up to 77% after multiple exposures. The fate of genetically modified cells from the different populations was followed through terminal differentiation using organotypic cultures. The epithelium of the organotypic cultures generated from each population exhibited both pseudostratified and stratified morphology, produced mucin, and stained positively with antibodies specific for basal and ciliated cells. The mucociliary epithelium also showed co-localization of these phenotypic markers with the expression of the vector-encoded beta-galactosidase gene. We conclude that retroviruses can efficiently transduce primary cultures of basal and secretory cells, and that both of these cell types can be progenitor cells of the airway epithelium. In vivo delivery of a retroviral vector containing a human placental alkaline phosphatase gene resulted in expression of the heterologous gene in rabbit tracheal epithelial cells. However, transduction efficiency was low and occurred only in the wounded trachea.

Successful gene therapy for the treatment of heritable or acquired diseases typically requires high efficiency gene transfer and sustained transgene expression. Indirect evidence on the basis of RNA analysis and in vivo competitive repopulation experiments in animal models suggests a correlation between transduction efficiency and the abundance of retrovirus receptors on the hematopoietic target cell. However, transduction by oncoretroviral vectors is also subject to other factors such as target cell cycle status and the composition of the virus-containing medium, making it difficult to determine the level of receptor expression required for efficient transduction. In the present study we investigated the impact of receptor expression level on transduction by a vector with a gibbon ape leukemia virus (GALV) envelope protein in a tetracycline-inducible tissue culture model that allowed for the cell cycle-independent, regulated expression of the GALV receptor (Pit1) in otherwise non-susceptible NIH 3T3 cells. Up-regulation of receptor RNA expression by 4.5-fold resulted in a mean 150-fold increase in transduction efficiency. We then analyzed cell surface expression of the Pit1 receptor using a fusion protein consisting of GALV SU portion of the viral envelope protein linked to the human IgG Fc. These experiments showed that tetracycline-regulated receptor induction resulted in a dose-dependent increase in binding of fusion protein. At maximum induction fusion protein binding increased up to five-fold which paralleled the increase in RNA expression, and correlated with the improved transduction efficiency. Finally, studies of pseudotype-specific fusion protein binding to human CD34-enriched cells revealed increased expression of retrovirus receptors after cytokine stimulation, although overall receptor expression in CD34(+)cells remained lower than in fibroblast cell lines efficiently transduced by amphotropic and GALV vectors.

XMRV (xenotropic murine leukemia virus-related virus) was initially discovered in association with prostate cancer and later with chronic fatigue syndrome (CFS). Its association with CFS is now largely discredited, and current results support a laboratory origin for XMRV with no reproducible evidence for infection of humans. However, some results indicating the presence of XMRV in prostate cancer are difficult to attribute to sample contamination. Here we have sought biological evidence that might confirm the presence of XMRV in prostate cancer samples previously having tested positive. We have tested for infectious XMRV and neutralizing antibodies against XMRV in blood plasma from 29 subjects with prostate cancer, and for infectious XMRV in prostate secretions from another five prostate cancer subjects. Nine of these subjects had previously tested positive for XMRV by PCR or by virus assay. We did not detect XMRV or related retroviruses in any sample, and the neutralizing activities of the plasma samples were all very low, a result inconsistent with XMRV infection of the plasma donors. We find no evidence for XMRV infection of any human subject tested, either by assay for infectious virus or for neutralizing antibodies. Our results are consistent with the majority of published studies on XMRV, which find that XMRV is not present in humans. The observed low to undetectable XMRV neutralization by human plasma indicates a lack of innate restriction of XMRV replication by soluble factors in human blood.

... Titre du document / Document title. Use of retroviral vectors for gene transfer and expression. Auteur(s) / Author(s). MILLER AD (1) ; MILLER DG (1) ; GARCIA JV ; LYNCH CM ; Affiliation(s) du ou des auteurs / Author(s) Affiliation(s). (1) Fred Hutchinson cancer res. ...

DNA analysis of peripheral-blood leukocytes is routinely used to demonstrate mutations in the dystrophin gene in patients with Duchenne's or Becker's muscular dystrophy. In approximately 35 percent of patients, DNA studies are not informative; in these patients immunochemical analysis of a muscle-biopsy specimen can determine whether dystrophin, the protein product of the gene for Duchenne's dystrophy, is present at reduced levels or absent. DNA analysis can be performed in amniocytes or chorionic-villus cells to identify mutations of the dystrophic gene prenatally, but immunochemical testing for dystrophin cannot be performed because the protein is not expressed in these cells. To circumvent this limitation in prenatal diagnosis, we induced myogenesis in 21 cultures of skin fibroblasts, 49 amniocyte cultures, and 6 chorionic-villus cell cultures by infecting the cells with a retrovirus vector containing MyoD, a gene regulating myogenesis. Transfection of MyoD into cells that do not normally develop into muscle cells results in the production of a protein that switches on myogenesis. We performed immunocytochemical analysis for dystrophin in the MyoD-converted muscle cells. We found that 60 of 61 myotube cultures from subjects with no family history of Duchenne's dystrophy expressed dystrophin. Both myotube cultures from the two patients with Becker's dystrophy also expressed dystrophin, but all cultures from nine patients and two fetuses with Duchenne's dystrophy were dystrophin-deficient. Immunocytochemical analysis of dystrophin in genetically altered non-muscle cells is feasible and may be applicable to the prenatal and postnatal diagnosis of Duchenne's muscular dystrophy when conventional DNA analysis is not informative.

Adeno-associated virus (AAV) vectors are attractive tools for direct intralumenal arterial gene transfer in interventional cardiology or cardiovascular surgery, but clinical application has been constrained by poor gene expression in this setting. To improve arterial wall gene expression, a hybrid promoter consisting of a cytomegalovirus (CMV) immediate-early enhancer, a chicken beta-actin transcription start site, and a rabbit beta-globin intron (CAG promoter) was substituted for the Rous sarcoma virus (RSV) promoter in an AAV type 2 vector with an alkaline phosphatase (AP) reporter gene. Intralumenal transduction of rabbit carotid arteries by an AAV2 vector containing a CAG promoter resulted in gene expression in a mean of > or = 80% of the lumenal area at 14 days following exposure, compared to < or = 25% gene-expressing area with the RSV promoter-based control vector. The high prevalence of gene expression was maintained at 3, 7, 14, and 28 days. Importantly, in carotid arteries transduced with the CAG promoter, gene product expression was readily visible by the third day following transduction whereas gene expression was rarely seen before day 10 using the RSV promoter in the same animal model. On histology, AP gene expression was predominantly in vascular smooth muscle cells although some endothelial cell expression was also present. Substituting the CAG for the RSV promoter results in widespread gene expression, demonstrating efficient arterial wall transduction by AAV2 vectors. This finding plus the early time to gene expression hold promise for AAV vectors as agents for direct intralumenal arterial wall gene delivery during cardiovascular interventions.

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus found in association with human prostate cancer and chronic fatigue syndrome, although these associations are controversial. XMRV shows at most 94% identity to known mouse retroviruses. Here we used XMRV-specific PCR to search for a more closely related source of XMRV in mice. While we could not find a complete copy, we did find a 3,600-bp region of XMRV in an endogenous retrovirus present in NIH/3T3 cells. These results show that XMRV has clear ancestors in mice and highlight another possible source of contamination in PCR assays for XMRV.

Xenotropic murine leukemia virus-related virus (XMRV) was first identified in human prostate cancer tissue and was later found in a high percentage of humans with chronic fatigue syndrome (CFS). While exploring potential disease mechanisms, we found that XMRV infection induced apoptosis in SY5Y human neuroblastoma cells, suggesting a mechanism for the neuromuscular pathology seen in CFS. Several lines of evidence show that the cell entry receptor for XMRV, Xpr1, mediates this effect, and chemical cross-linking studies show that Xpr1 is associated with the Gβ subunit of the G-protein heterotrimer. The activation of adenylate cyclase rescued the cells from XMRV toxicity, indicating that toxicity resulted from reduced G-protein-mediated cyclic AMP (cAMP) signaling. Some proteins with similarity to Xpr1 are involved in phosphate uptake into cells, but we found no role of Xpr1 in phosphate uptake or its regulation. Our results indicate that Xpr1 is a novel, atypical G-protein-coupled rec...

Adeno-associated virus (AAV) vectors have been shown to preferentially transduce hepatocytes after systemic administration in adult mice and to provide long-term expression of introduced genes. One application of this technology would be for the production of granulocyte colony-stimulating factor (G-CSF), which increases mature neutrophil numbers in humans and in animals, and has therapeutic effects in disorders featuring chronic neutropenia, including cyclic, severe congenital, and idiopathic neutropenia, and glycogen storage disease type Ib. We have treated mice by tail vein injection of AAV vectors encoding human G-CSF, and have detected high G-CSF levels and marked elevation of neutrophil counts for at least 5 months. A therapeutically relevant amount of G-CSF production was obtained when the liver-specific mouse albumin promoter-enhancer was used to drive G-CSF expression. In mice receiving higher amounts of vector, plasma levels of human G-CSF gradually increased over 3 weeks to high concentrations, whereas for lower amounts human G-CSF remained at initial, low levels. The previously observed effect of gamma irradiation, to increase AAV transduction rates, was diminished when large amounts of vector were used. Absolute neutrophil counts increased 10- to 50-fold for the period of observation to levels that would be therapeutic in the treatment of cyclic neutropenia. In conclusion, gene therapy with AAV vectors synthesizing G-CSF shows promise for the treatment of disorders featuring neutropenia.

A variety of retroviral vectors for transduction and expression of clotting factor VIII (FVIII) were constructed by using truncated forms of a FVIII cDNA lacking part or all of the nonessential B-domain sequences. Both the titer of virus and FVIII protein production from the vectors was about 2 orders of magnitude lower than the virus titer and protein production from identical retroviral vectors containing other cDNAs, including clotting factor IX. These decreases could be entirely explained by an observed 100-fold lower accumulation of vector RNAs containing the FVIII sequences in comparison to vectors containing other cDNA sequences. Deletion analysis of one of the FVIII vectors demonstrated that diffuse sequences within the FVIII coding region had a deleterious effect upon vector titer and RNA accumulation. One inhibitory signal could be localized to a 1.2-kb stretch of DNA, but further localization was not possible as additional size reduction abolished the activity. These results indicate that expression of FVIII is regulated by signals within FVIII coding sequence that result in decreased RNA accumulation and FVIII protein production. Alteration of these inhibitory signals to permit high-level FVIII production may be difficult due to the wide distribution of these signals within the coding region of the protein.

In 1991 we reported gene transduction into autologous long-term repopulating marrow cells in dogs using amphotropic helper-free retrovirus vectors containing the bacterial neomycin phosphotransferase gene (neo) and the human adenosine deaminase gene (ADA). Two of the dogs are still alive and healthy now more than 5 years after transplantation of transduced autologous marrow cells. In one of the surviving dogs, polymerase chain reaction (PCR) analysis showed the neo and ADA genes to be present in peripheral blood granulocytes and lymphocytes up to the present time. The estimated percentage of neo-positive cells ranged from < 0.001% to 0.1%. ADA mRNA expression was detected by reverse transcriptase PCR (RT-PCR) in granulocytes 63 months after transplantation. The other surviving dog failed to show either persistence or expression of the transduced genes after 50 months. Three additional dogs have been transplanted according to the same transduction protocols and with the same retrovirus vectors, and persistence of the transduced neo gene has been documented in peripheral blood myeloid and lymphoid cells along with G418-resistant colony-forming unit-granulocyte/macrophage (CFU-GM) for now more than 2 years. These findings represent the longest follow-up of retrovirus-mediated gene transduction in any animal species. Long-term transduction efficiency, though, has remained low and will need to be improved for therapeutic application to be possible.

The transduction efficiency of adeno-associated virus (AAV) vectors in various somatic tissues is determined primarily by the viral capsid proteins. In contrast to vectors made with AAV type 2 capsids, those having type 5 or 6 capsids show high transduction rates in airway epithelial cells, in a range that should be sufficient for treating lung disease. Here we have compared the properties of vectors made with AAV5 or AAV6 capsid proteins to determine whether their receptor usage is similar, and found several differences between the viruses. First, an AAV6 vector did not hemagglutinate red blood cells, whereas an AAV5 vector did, and this property was sialic acid dependent. Second, AAV5 vector transduction required sialic acid in all cells tested, whereas AAV6 vector transduction was sialic acid dependent or independent, depending on the target cells tested. Third, levels of an AAV6 vector that interfered with entry of another AAV6 vector only poorly inhibited AAV5 vector transduction and vice versa. These results indicate that AAV5 and AAV6 vectors use distinct cellular receptors for cell entry. Although both AAV5 and AAV6 vectors exhibited high transduction rates in well-differentiated human airway epithelial cultures, they exhibited distinct cell-type transduction profiles in mouse lung that may reflect differences in receptor usage.

Clinical use of human granulocyte-colony stimulating factor (hG-CSF) to treat various diseases involving neutropenia has been previously shown to (1) successfully increase circulating neutrophils, (2) reduce condition-related infections, and (3) cause few side effects in patients. To alleviate the symptoms of neutropenia, the patient must receive frequent injections of recombinant hG-CSF. Permanent ways to deliver stable levels of the molecule to the patient are being investigated. Among them, the transplantation of hG-CSF-secreting cells has been proposed and performed successfully in rodents, using fibroblast cell lines and primary muscle cells. We thus investigated whether similar results could be obtained by intramuscular myoblast transplantation in a large animal model. When 1-3 x 10(8) myoblasts were injected into three Macaca mulatta, hG-CSF was detected at high levels (300-900 pg/ml), which in turn led to a four- to fivefold increase in circulating neutrophils. However, both the concentrations of hG-CSF and neutrophil levels were found to decrease over time. Nonetheless, neutrophils were found at higher levels from the fourth week until the end the experiment (up to 29 weeks) in G-CSF monkeys compared with control animals. These results show that transplantation of hG-CSF-secreting myoblasts may indeed be a therapeutic option for the treatment of neutropenic patients.

Protein expression from retroviral vectors is often highest when the expressed cDNA is driven by the retroviral promoter. However, the typical retroviral vector design places the cDNA downstream of the retroviral packaging signal and far from the retroviral promoter. In an attempt to improve protein production levels from cDNAs expressed in retroviral vectors, we inserted the MyoD or the purine nucleoside phosphorylase (PNP) cDNAs into the R regions of both retroviral LTRs, close to the retroviral promoter and just upstream of the polyadenylation signal present in each long terminal repeat (LTR). These R-region double-copy vectors could be produced in unrearranged form, although the titer was about seven-fold lower than that of typical vectors. R-region positioning of the MyoD cDNA resulted in five-fold higher MyoD expression compared to MyoD expression in a typical vector, whereas PNP expression was not improved. Thus, R-region double-copy vectors provide an alternative vector design that can improve protein expression from some cDNAs.

Gene therapy of the lung requires the introduction and expression of a therapeutic gene in airway cells. Although retroviral vectors may be useful in this context, the ability of retroviruses to infect specific cell types in the airway is not known. In this study, we examined the ability of amphotropic recombinant retroviral vectors to transduce primary cultures of rabbit airway epithelial cell populations purified for basal or secretory cells. Transduction efficiencies in basal and secretory cell populations were found to be similar; about 27% after a single exposure to vector, and up to 77% after multiple exposures. The fate of genetically modified cells from the different populations was followed through terminal differentiation using organotypic cultures. The epithelium of the organotypic cultures generated from each population exhibited both pseudostratified and stratified morphology, produced mucin, and stained positively with antibodies specific for basal and ciliated cells. The mucociliary epithelium also showed co-localization of these phenotypic markers with the expression of the vector-encoded beta-galactosidase gene. We conclude that retroviruses can efficiently transduce primary cultures of basal and secretory cells, and that both of these cell types can be progenitor cells of the airway epithelium. In vivo delivery of a retroviral vector containing a human placental alkaline phosphatase gene resulted in expression of the heterologous gene in rabbit tracheal epithelial cells. However, transduction efficiency was low and occurred only in the wounded trachea.

Gene transfer to skeletal muscle was examined as a means of gene therapy for neutropenia. A recombinant retrovirus containing a human granulocyte colony-stimulating factor (G-CSF) gene was introduced into primary human or rat myoblasts, which were then shown to produce biologically active G-CSF. Transplantation of G-CSF-producing rat myoblasts into the muscle of syngeneic rats resulted in a 15-fold increase in absolute neutrophil counts. This increase correlated with detection of circulating human G-CSF protein throughout the 6-month duration of the experiment. These results clearly demonstrate long-term production of therapeutically relevant amounts of a human protein by normal cells in vivo.