Lentiviral protein delivery of meganucleases in human cells mediates gene targeting and alleviates toxicity.

Site-specific endonucleases can be engineered for custom recognition of any genetic locus and used for gene targeting. Yet, the prolonged expression and accumulation of these nucleases in cells lead to toxic effect. Here we describe an efficient and quantitative method for introducing nucleases into cells as proteins packaged within lentiviral vector particles. I-CreI-derived meganucleases, which can be engineered as single-chain proteins, were incorporated into lentiviral vector particles either without modification or as fusions with cyclophilin A. The small amount of nuclease delivered by the viral particles is sufficient to induce efficient targeted mutagenesis in human HEK293H and primary T cells. When a repair template sequence was packaged in the lentiviral vector, high levels of homologous gene targeting were obtained and toxicity was markedly reduced.

Correction of lysosomal storage in the liver and spleen of MPS VII mice by implantation of genetically modified skin fibroblasts.

Genetic defects of lysosomal hydrolases result in severe storage diseases and treatments based on enzyme replacement have been proposed. In mice lacking beta-glucuronidase, which develop a disease homologous to human mucopolysaccharidosis type VII (Sly syndrome), we have used autologous implants of genetically-modified skin fibroblasts for the continuous in vivo production of the enzyme. The human beta-glucuronidase cDNA was introduced with a retroviral vector into mutant mice skin fibroblasts grown in primary culture. Fourteen mutant mice were implanted intraperitoneally with these modified cells embedded into collagen lattices. All animals expressed beta-glucuronidase from the vascularized neo-organs that developed after implantation and accumulated the enzyme in their tissues. A complete disappearance of the lysosomal storage lesions was observed in their liver and spleen.

Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors.

Haemophilia B, or factor IX deficiency, is a X-linked recessive disorder that occurs in about one in 25,000 males, and severely affected people are at risk for spontaneous bleeding into numerous organs. Bleeding can be life-threatening or lead to chronic disabilities with haemophilic arthropathy. The severity of the bleeding tendency varies among patients and is related to the concentration of functional plasma factor IX. Patients with 5-30% of the normal factor IX have mild haemophilia that may not be recognized until adulthood or after heavy trauma or surgery. Therapy for acute bleeding consists of the transfusion of clotting-factor concentrates prepared from human blood and recombinant clotting factors that are currently in clinical trials. Both recombinant retroviral and adenoviral vectors have successfully transferred factor IX cDNA into the livers of dogs with haemophilia B. Recombinant retroviral-mediated gene transfer results in persistent yet subtherapeutic concentrations of factor IX and requires the stimulation of hepatocyte replication before vector administration. Recombinant adenoviral vectors can temporarily cure the coagulation defect in the canine haemophilia B model; however, an immune response directed against viral gene products made by the vector results in toxicity and limited gene expression. The use of recombinant adeno-associated virus (rAAV) vectors is promising because the vector contains no viral genes and can transduce non-dividing cells. The efficacy of in vivo transduction of non-dividing cells has been demonstrated in a wide variety of tissues. In this report, we describe the successful transduction of the liver in vivo using r-AAV vectors delivered as a single administration to mice and demonstrate that persistent, curative concentrations of functional human factor IX can be achieved using wild-type-free and adenovirus-free rAAV vectors. This demonstrates the potential of treating haemophilia B by gene therapy at the natural site of factor IX production.

Long-term delivery of a lysosomal enzyme by genetically modified fibroblasts in dogs.

We have evaluated the feasibility and efficacy of intraperitoneal implants (neo-organs) for protein delivery in large animals. Skin biopsies were taken from four healthy dogs. Primary fibroblast cultures were transduced with a retroviral vector coding for the human beta-glucuronidase. One to six lattices each containing 10(9) skin fibroblasts were implanted into the omentum of the donor animal. Laparotomies performed at regular intervals showed vascularized neo-organs without local inflammation. Human beta-glucuronidase levels equivalent to 0.8 to 3.1% of the endogenous canine activity were detected for up to 340 days on liver biopsy samples. These results indicate that neo-organs can be considered for the long-term delivery of therapeutic proteins or enzymes in humans.

AAV vectors for RNA-based modulation of gene expression.

At the post-transcriptional level, gene expression is largely regulated through a network of molecular machines that regulate pre-mRNA maturation integrity, transport, translation and degradation. These processes are based on the formation of nucleoprotein complexes and require the recognition of sequence motifs on the RNA. By masking these targets with complementary RNA sequences forming Watson-Crick base pairing, it is possible to efficiently and specifically impact on the cell phenotype, or to compensate the deleterious effect of mutations. Here we review how the adeno-associated virus technology is being exploited for expressing non-coding RNAs in tissues such as the brain, muscle or liver, in functional genomic studies as well as for the development of novel therapeutic strategies.

Efficient gene transfer in skeletal muscle with AAV-derived bicistronic vector using the FGF-1 IRES.

IRESs (internal ribosome entry sites) are RNA elements behaving as translational enhancers in conditions of global translation blockade. IRESs are also useful in biotechnological applications as they allow expression of several genes from a single mRNA. Up to now, most IRES-containing vectors use the IRES from encephalomyocarditis virus (EMCV), highly active in transiently transfected cells but long and not flexible in its positioning relative to the gene of interest. In contrast, several IRESs identified in cellular mRNAs are short and flexible and may therefore be advantageous in gene transfer vectors such as those derived from the adeno-associated virus (AAV), where the size of the transgene expression cassette is limited. Here, we have tested bicistronic AAV-derived vectors expressing two luciferase genes separated by the EMCV- or fibroblast growth factor 1 (FGF-1) IRES. We demonstrate that the AAV vector with the FGF-1 IRES, when administrated into the mouse muscle, leads to efficient expression of both transgenes with a stable stoechiometry, for at least 120 days. Interestingly, the bicistronic mRNA containing the FGF-1 IRES leads to transgene expression 10 times superior to that observed with EMCV, in vivo. AAV vectors featuring the FGF-1 IRES may thus be advantageous for gene therapy approaches in skeletal muscle involving coexpression of genes of interest.

Enhanced lentiviral transduction of monocyte-derived dendritic cells in the presence of conditioned medium from dying monocytes.

Lentiviral vectors (LVs) are attractive vehicles for the transduction of human dendritic cells (DCs) in order to mobilize their endogenous antigen presentation pathways. We analyzed here how to improve the efficiency of LV transduction, which we performed at the initial stages of the differentiation of purified monocytes into dendritic cells (Mo-DCs). Using LVs pseudotyped with the vesicular stomatitis virus envelope G glycoprotein (VSV-G), we found that a conditioned medium derived from dying monocytes (MCM) improved by 2- to 10- fold the proportion of transduced Mo-DCs. This enhanced transduction efficiency requires the presence of MCM during the initial stage of LV transduction and does not affect the phenotype and antigen presentation function of terminally differentiated Mo-DCs. Importantly, we found that MCM derived from a human acute monocytic leukemia cell line, THP-1, was equally effective. The MCM activity was heat stable (56 degrees C) and was present in the soluble fraction after high-speed centrifugation. Altogether our results show that a soluble factor present in dying monocyte cultures can replace advantageously facilitating agents such as Polybrene, to achieve high LV transductions levels. This protocol can be performed with autologous monocytes and is therefore applicable in clinical settings.

Regulation of gene expression in vivo following transduction by two separate rAAV vectors.

Control of gene expression is important to gene therapy for purposes of both dosing and safety. In vivo regulation of gene expression was demonstrated following co-injection of two separate recombinant adeno-associated virus vectors, one encoding an inducible murine erythropoietin transgene and the other a transcriptional activator, directly into the skeletal muscle of adult immunocompetent mice. Transcription was controlled by systemic administration or withdrawal of tetracycline over an 18 week period, demonstrating that the two vectors were capable of transducing the same cell. Cellular or humoral immune responses against the transactivator protein were not detected.

Adeno-associated virus pseudotype 5 vector improves gene transfer in arthritic joints.

The potential for gene delivery to joints, using recombinant adeno-associated virus (rAAV) vectors for the treatment of rheumatoid arthritis (RA), has received much attention. Different serotypes have different virion shell proteins and, as a consequence, vary in their tropism for diverse tissues. The aim of this study was to compare the transduction efficiency of different AAV serotypes encoding murine secreted alkaline phosphatase (mSEAP) or Escherichia coli beta-galactosidase for intraarticular gene delivery in an experimental model of arthritis. The vectors contained AAV2 terminal repeats flanking the reporter gene in an AAV1, AAV2, or AAV5 capsid, producing the pseudotypes rAAV-2/1, rAAV-2/2, and rAAV-2/5. Left knee joints of mice with collagen-induced arthritis were injected and transgene expression was analyzed by chemiluminescence or direct in situ staining of frozen sections. We show for the first time that intraarticular gene transfer with AAV- 2/5 was far more efficient than with the other serotypes tested. Transgene expression was detectable as early as 7 days after injection, reached a maximum at 21 days, and was stably expressed for at least 130 days, whereas AAV-2/1- and AAV-2/2-mediated expression levels were barely detectable. These findings provide a practical application for future local AAV-mediated gene therapy trials in RA.

Nucleotide sequence and comparative analysis of the human papillomavirus type 18 genome. Phylogeny of papillomaviruses and repeated structure of the E6 and E7 gene products.

The complete nucleotide sequence and genomic organization of human papillomavirus type 18, associated with cervical cancer, has been established. A detailed comparative analysis was undertaken leading to the identification of a number of features specific for genital papillomaviruses and the construction of a phylogenetic tree. Genital papillomaviruses differ from other human and animal papillomaviruses as they possess a longer E1 open reading frame (ORF) and have a characteristic control region. Phylogenetically, HPV 18 is located between the benign genital viruses, HPV 6 and HPV 11, and the malignant isolates, HPV 16 and HPV 33, and may represent an evolutionary intermediate among oncogenic papillomaviruses. Viral gene products known to be involved in cellular transformation are those of ORFs E5, E6 and E7. Significant sequence variation was found between the E6 to E7 regions of different integrated forms of HPV 18. On re-examination of the E6 primary structures we noticed that the gene has evolved by successive duplications of a unit encoding 33 amino acids, which include a Cys-X-X-Cys motif. Furthermore, the E7 gene product has apparently evolved in the same manner and is related to E6. Both gene products bear a striking resemblance to the transcriptional factor IIIA of Xenopus laevis, the prototype of a new class of nucleic acid binding proteins.

Regulation of diaminopimelate decarboxylase synthesis in Escherichia coli. II. Nucleotide sequence of the lysA gene and its regulatory region.

The complete nucleotide sequence of the lysA gene and its regulatory region was determined. At the 3' end of the lysA gene an open reading frame was revealed in the opposite direction and was identified as the galR coding region. Only six base-pairs are present between the two translational stops and thus both transcription units are overlapping in vivo. Translational gene fusions constructed in vitro with the beta-galactosidase gene were used to identify the lysA initiating ATG. The sequence encodes a 420 amino acid long peptide for a predicted molecular weight of 46,099. No attenuation-like sequence was found at the beginning of the lysA gene. A target of the LysR activator protein was localized on a 73 base-pair fragment found 48 base-pairs upstream from the lysA coding region. The presence of this DNA sequence on a multicopy plasmid led to a net decrease of lysA expression, indicating limiting amounts of active LysR protein in the cytoplasm.

Retroviral vectors for the transduction of the PML-RARalpha fusion product of acute promyelocytic leukemia.

We have designed several retroviral constructs to transduce the PML-RA Ralpha fusion product of human acute promyelocytic leukemia. Our aim to generate high-titer stable vector-producing cell lines was hindered by a toxic effect of PML-RARalpha expression on packaging cells. To circumvent this, we tested retroviral vectors expressing the transgene from several internal promoters including inducible and myelospecific promoters. To compare efficiency of these constructs in their ability to generate protein expression in the appropriate target cells and optimal viral titers, we used the BOSC23 transient packaging cell line. We found that the direct-oriented vector did not ensure tissue-specificity of PML-RARalpha expression, while the reverse-oriented retroviral vector did. The latter construct, however, failed to generate high-titer recombinant virus. This study exemplifies the unpredictable behavior of retroviral constructs and the superiority of transient systems for transduction of a toxic product.

Correction of deficient enzyme activity in a lysosomal storage disease, aspartylglucosaminuria, by enzyme replacement and retroviral gene transfer.

The ability of lysosomal enzymes to be secreted and subsequently captured by adjacent cells provides an excellent basis for investigating different therapy strategies in lysosomal storage disorders. Aspartylglucosaminuria (AGU) is caused by deficiency of aspartylglucosaminidase (AGA) leading to interruption of the ordered breakdown of glycoproteins in lysosomes. As a consequence of the disturbed glycoprotein catabolism, patients with AGU exhibit severe cell dysfunction especially in the central nervous system (CNS). The uniform phenotype observed in these patients will make effective evaluation of treatment trials feasible in future. Here we have used fibroblasts and lymphoblasts from AGU patients and murine neural cell lines as targets to evaluate in vitro the feasibility of enzyme replacement and gene therapy in the treatment of this disorder. Complete correction of the enzyme deficiency was obtained both with recombinant AGA enzyme purified from CHO-K1 cells and with retrovirus-mediated transfer of the AGA gene. Furthermore, we were able to demonstrate enzyme correction by cell-to-cell interaction of transduced and nontransduced cells.

Long-term secretion of therapeutic proteins from genetically modified skeletal muscles.

Protein delivery from genetically modified skeletal muscle has been reported previously. However, a stable and prolonged secretion was obtained in immunocompromised or newborn animals only. To evaluate the clinical relevance of this approach, we have transduced myoblasts from an adult beta-glucuronidase-deficient (MPS VII) mouse with retroviral vectors carrying either the human beta-glucuronidase cDNA or the murine erythropoietin (Epo) cDNA. The cells were then grafted into the tibialis anterior muscle of adult immunocompetent MPS VII recipients. Protein expression was controlled either by ubiquitous or muscle-specific transcriptional regulatory elements. Animals were analyzed over an 8-month period. The in situ detection of beta-glucuronidase activity revealed up to 60% of genetically modified myofibers in the recipient muscles. The human desmin promoter and enhancer showed the highest in vivo expression. Secretion of beta-glucuronidase induced a disappearance of lysosomal storage lesions in the liver and spleen of recipient animals. Delivery of Epo led to a permanent increase of hematocrit values over 3 months. These results showed that the transplantation of genetically modified myoblasts allowed a sustained secretion of recombinant proteins at therapeutic levels in immunocompetent adult mice. They suggest that the approach may be considered for human applications.

In situ retrovirus-mediated gene transfer into dog liver.

Dogs were used as a large animal model to assess the feasibility and safety of a surgical method for gene transfer into hepatocytes in vivo. This method, which we previously described in rats, consists of a partial hepatectomy aimed at inducing liver regeneration, followed by the selective in situ perfusion of the remnant liver parenchyma with a retrovirus preparation. Isolation of the liver was obtained by clamping the afferent and efferent blood vessels, a procedure that prevented retroviral vector dissemination and genetic modification of nonhepatic organs. A helper-free retrovirus vector encoding beta-galactosidase targeted to the nucleus was perfused in the liver of 5 golden retriever dogs. Volumes up to 1,650 ml of fresh or concentrated vector stocks were perfused and the procedure was well tolerated. Gene transfer, observed in 3 of 5 treated dogs when documented on liver biopsy fragments obtained at day 4, involved 0.15-0.6% hepatocytes and persisted at equivalent levels at the time of sacrifice, 6 weeks later. No propagation of the vector to other tissues was detected. These observations suggest that the selective perfusion of the regenerating liver might be considered an alternative to liver transplantation for the treatment of certain severe genetic liver disorders, or for the delivery of a therapeutic protein into the serum.

In vivo delivery of human alpha-L-iduronidase in mice implanted with neo-organs.

Mucopolysaccharidose type I is a lysosomal storage disease caused by a deficiency in the enzyme alpha-L-iduronidase (IDUA). The existence of a secretory pathway for lysosomal enzymes and the capture of secreted molecules by distant cells through binding to mannose-6-phosphate receptors have provided a rationale for enzyme replacement therapy in lysosomal storage diseases. We have used genetically modified fibroblasts implanted into neo-organs as an in vivo delivery system for IDUA. The human IDUA cDNA was isolated and inserted into a retroviral vector where it was expressed from the phosphoglycerate kinase 1 gene promoter. MPS I fibroblasts transduced with this vector showed high levels of IDUA activity and secreted phosphorylated molecules that could be internalized by naive deficient cells. Neo-organs containing 2 x 10(7) IDUA-secreting cells were implanted into nude mice. Human and murine IDUA activities were measured in the liver and spleen of animals sacrificed 35-77 days after implantation. Human IDUA activity corresponded to 0.6-2.3% of the murine enzyme activity in the liver and to 0.1-0.3% in the spleen. These data indicated that human IDUA was secreted from neo-organs and internalized in distant tissues.

Efficient and stable adeno-associated virus-mediated transduction in the skeletal muscle of adult immunocompetent mice.

Recombinant adeno-associated virus (rAAV) vectors were evaluated for gene transfer into the skeletal muscle of adult immunocompetent mice. A study using a vector encoding nuclear localized beta-galactosidase (rAAV-nls-lacZ) examined: (i) the efficiency and duration of transgene expression; (ii) the status of the AAV genome in the transduced fibers; and (iii) the possibility of improving gene transfer by inducing muscle regeneration. In the absence of regeneration, the injection of 1.7 x 10(7) particles in the quadriceps resulted in gene transfer to 10-70% of myofibers. Histological analysis indicated that the vector was able to reach myofiber nuclei distant from the injection point. Cellular infiltrates were absent at early time points but became conspicuous in the vicinity of some positive fibers at 4-8 weeks and subsided by 26 weeks. Southern analysis indicated that one to three copies of the vector genome were present per cell genome equivalent. They were associated with high-molecular-weight DNA in the form of tandem oligomers or interlocked circles. Gene transfer was not facilitated in the regenerating muscle. Rather, an early inflammatory response resulted in the elimination of most positive fibers after 8 weeks. The presence of regenerated fibers with beta-galactosidase-positive nuclei suggested that myoblasts had been transduced and were able to fuse to form new fibers. Gene transfer in the absence of immune reactions against the transgene product was studied by injecting mice with a rAAV carrying the murine erythropoietin (mEpo) cDNA. Dose-dependent elevation in the hematocrit was measured for over 200 days and corresponded to 5- to 20-fold increases in plasma Epo levels. We conclude that AAV vectors efficiently and stably transduce post-mitotic muscle fibers and myoblasts in vivo.

Retroviral particles produced from a stable human-derived packaging cell line transduce target cells with very high efficiencies.

The goal of this work was to determine whether a stable 293 amphotropic packaging line, which we have designated 293-SPA, is useful for the production of high-titer stable virus by comparison to the murine psiCRIP line. Here, we report our unexpected findings that particles derived from the 293-SPA line transduce target cells (both NIH-3T3 cells and primary melanoma cells) with greatly enhanced efficiencies (at least 10-fold) compared to particles derived from the psiCRIP packaging line. We show that the presence of a transferable inhibitor in the psiCRIP line at least partially accounts for this dramatic difference in transduction efficiency. This work has important implications for improving the efficiency of retrovirus-mediated gene transfer in general as well as in the design of new packaging cell lines.

Functional retroviral vector for gene therapy of xeroderma pigmentosum group D patients.

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder characterized by an increased frequency of skin cancer following minimal sunlight exposure. Cells isolated from XP patients are also hypersensitive to UV rays and UV-like chemicals. This sensitivity is directly related to a defect in the early steps of nucleotide excision repair (NER) of damaged DNA. No efficient treatment is available for this disease and skin cancer prevention can only be achieved by strict avoidance of sunlight exposure. Thus, we are developing a model for gene therapy in XP, particularly for patients belonging to group D. We report here the construction of a retroviral vector (LXPDSN) containing the XPD (ERCC2) cDNA, which fully complements the DNA repair deficiency of primary skin fibroblasts. Efficient integration, mRNA synthesis, and protein expression of the XPD gene were obtained in all LXPDSN-transduced XP-D fibroblasts tested. Full correction of the DNA repair defect was observed with all DNA repair assays used, such as an increased survival after UV-radiation of the transduced cells, a normal level of DNA repair synthesis (UDS), and the reactivation of a UV-irradiated reporter vector. This retroviral vector will be used to modify keratinocytes genetically to produce repair proficient reconstituted skin for engraftment to XP patients.

Papillomavirus genomes: sequences and consequences.

The complete nucleotide sequences of four papillomavirus genomes recently determined (human papillomavirus types 1a and 6b, bovine papillomavirus type 1, and the Shope cottontail rabbit papillomavirus) shed light on the molecular genetics of these epitheliotropic viruses. The organization of the four genomes is very similar, and their comparative analysis suggests new hypotheses about the nature of the viral proteins, their involvement in virus structure, and their interaction with the host cell, the terminally differentiating keratinocyte. It also allows us to consider, at the molecular level, the problem of papillomaviruses' host range and evolutionary relationship.