Principe et applications thérapeutiques du transfert de gène
1. Targeted integration of transgenes
Successful gene therapy treatments using retroviral vectors have revealed a genotoxicity associated with the insertion of a therapeutic transgene into the patient cells genome [1]. These clinical observations have called for a better control of the integration process. Our goal is to use homologous recombination (HR) to drive integration of a sequence carried by lentiviral vectors into a precise and unique site on the human genome. For this, we are building lentivirus based vector systems that bring into the cell a site specific endonuclease and a recombination matrix with homologies to the targeted locus. The meganuclease creates a double stranded break at a selected chromosomal site. This is expected to significantly enhance recombination with the matrix present on the vector. These studies are performed in collaboration with Cellectis SA, a company that develops custom-made meganucleases for genome editing.
We have shown that highly efficient HR is obtained when the nuclease is delivered as a protein incorporated in the lentiviral particles. I-SceI, a prototypic meganuclease from yeast, was incorporated into the virions as a fusion with Vpr, an HIV accessory protein. Non-integrating lentiviral vectors (NILV) containing a recombination matrix and the I-SceI fusion protein were produced at high titers. We have used a CHO-derived reporter cell line in which I-SceI mediated HR events result in the repair of a puromycin resistance gene. These cells were transduced by NILVs containing both I-SceI as a Vpr fusion and the recombination matrix, or by separate vectors encoding either the recombination matrix or I-SceI. Puromycin resistant cell clones were selected and analysed by PCR and Southern blot for targeting events. A majority of puromycin resistant clones contained the expected genomic structure and globally, our data indicate that the transfer of the endonuclease as a virion incorporated protein results in up to 5% of HR events, in a dose dependent manner. HR levels were lower when the
fusion protein and the recombination matrix were provided in separate viral particles, suggesting that the physical association of the two in the same lentiviral pre-integration complex may be important for efficient targeting. Interestingly, the levels of HR obtained when I-SceI was encoded by the lentiviral vector were consistently lower. Finally, we have shown that Vpr fusions could also be used for the transduction of a single chain meganuclease (scMN) engineered from I-CreI with a recognition site in the human Rag-1 gene.
Our current objectives are to define and characterize safe chromosomal loci for the integration of therapeutic transgenes in human cells as well as optimal systems for delivering Meganucleases in vivo using viral vectors. A system for targeted integration will be a significant improvement over existing tools for therapeutic gene delivery using viral vectors. Beyond this, the possibility to deliver meganucleases would enable to use them in other therapeutic applications, for instance for inactivating DNA viruses (Hepatitis B, Papillomaviruses, Cytomegaloviruses, etc…) or targeting dominant mutations in genetic and acquired disorders.
2. Interventions on mRNAs
Gene expression is largely regulated at the post-transcriptional level through a network of molecular machines that ensure pre-mRNA splicing, transport and translation [2]. A variety of mRNAs with different exon composition, stability or sub-cellular localisation can be produced from a given transcription unit. This is a central mechanism for generating diversity from the genome information as illustrated in the brain and in the immune system. In this context, it is not surprising that up to 50% of disease-associated mutations in humans affect the processing of pre-mRNAs, by inactivating splicing signals, creating cryptic ones or modifying binding sites for splicing regulators [3]. These mutations results in exon skipping, inclusion of cryptic exons or intron retention, and to the production of non-functional mRNAs or proteins. In addition, neutral exonic mutations that disrupt splicing enhancers and silencers may have an important impact on the delicate balance of alternative splicing, which is observed in up to 75% of transcription units in the human genome [4].
The modulation of the pre-mRNA maturation processes, in particular spliceosome assembly and activity is likely to be a powerful mode of impacting on a cellular phenotype and compensating the deleterious effect of mutations [5]. It can be achieved with a high specificity, using antisense oligoribonucleotides (AON) that mask key determinants of splicing through Watson-Crick pairing with the pre-mRNA. Successful AON-mediated restoration of correct splicing has been reported with a variety of mutations associate with genetic diseases. AON masking of an Intronic Splicing Silencer (ISS) in the Smn2 gene enhances exon 7 inclusion and suggests a therapeutic approach for Spinal Muscular Atrophy [6]. In cases where splicing regulatory sequences have been altered by mutation, a functional sequence can be brought back in trans by linking it to an anchoring AON (tailed RNA) [7]. Finally, AONs that mask splicing signals can be exploited to induce exon-skipping and remove deleterious mutations or re-establish a reading frame disrupted by a deletion. Yet, only proteins in which the domain corresponding to the skipped exon can be removed without damaging the function are relevant here. It is the case of Dystrophin, the protein affected in Duchenne Muscular Dystrophy, Collagen VII involved in Dystrophic Epidermolysis Bullosa, and LEKTI (lympho-epithelial Kazal-type-related inhibitor) in Netherton Syndrome . We have previously adapted viral gene transfer vector for the delivery of antisense sequences and demonstrated their efficacy for mRNA based intervention in the context of Duchenne Muscular Dystrophy. Therapeutic exon-skipping was obtained in the skeletal muscle of mice [8] and dogs affected with the disease and in cells from human patients [9], when the appropriate antisense sequences were linked to a modified U7 small nuclear RNA (snRNA), which was itself expressed from Adeno-associated or lentiviral vectors. Our current goal is to expend these findings and further develop the technology towards applications to other pathologies. This program is expected to lead to a better understanding of the biology of antisens mediated modulation
3. Mechanisms of viral infectivity enhancement by HIV-1 Nef
We have a long standing interest for understanding the mechanisms of cellular permissivity to viral infection. In the case of HIV, viral accessory proteins play a major role in tuning host-virus interactions and the characterization of their cellular partners illuminates the mechanisms of viral adaptation. Our research focuses on the ability of the viral accessory protein Nef to increase virus infectivity. Wild-type HIV induces AIDS in humans, while viruses devoid of nef gene are highly attenuated. Yet, under certain circumstances, Nef does not increase virus infectivity, suggesting that its activity requires an interaction with other partners. We have shown that Nef acts during virions biogenesis to increase HIV-1 infectivity. The “Nef effect” is dependent on the virion envelope glycoprotein (Env) but Nef does not affect fusion. Based on this premises, we are trying to understand
whether virions acquire the infectivity enhancement in a particular cellular compartment. and how Nef affects the incorporation or exclusion of cellular factors into or from viruses,
4. Towards a gene therapy clinical trial for the treatment of Sanfilippo type A syndrome.
Sanfilippo syndrome or mucopolysaccharidosis type III (MPSIII,) is a lysosomal storage disease in which an autosomal recessive genetic defect results in the accumulation of partially degraded oligosaccharides of heparan sulfate. There are four biochemical subtypes of Sanfilippo syndrome with similar clinical manifestations. Affected children progressively develop profound mental retardation without the severe skeletal involvement described in other MPS. Mutations in the gene encoding N-sulfoglycosamine sulphohydrolase (SGSH) are responsible for MPSIIIA which represent about two third of the Sanfilippo cases. There is currently no available treatment for the Sanfilippo syndrome but major advances have been obtained over the past ten years, demonstrating the feasibility of AAV-mediated stable gene transfer into the brain resulting in extended intra-cerebral distribution of enzyme in animal models of Sanfilippo and correction of the major clinical manifestation [10]. Under the sponsorship of Alliance Sanfilippo, we coordinate the preparation of a clinical trial for the treatment of Sanfilippo type A syndrome by intracerebral administration of an AAV2.10 vector encoding the human Sulfamidase cDNA. The vector has been designed and validated in MPSIIIA mice in collaboration with the team of Andrea Ballabio (Tigem, Naples) and Jean Michel Heard (Institut Pasteur) and John Hopwood (Adelaïde Australia). It has been manufactured under GMP for clinical use and tested in pharmacological toxicology studies. The clinical trial will be under the responsibility of Marc Tardieu (Hôpital Bicêtre) in collaboration with Michel Zerah (Neurochirurgie, Necker), and is expected to start in the last quarter of 2010.
5. Gene Transfer Vector Core
(Institut Fédératif de Recherche Necker Enfants Malades, Université Paris Descartes, Fondation Imagine)
Our interactions with other laboratories on the campus have convinced us early on that a core facility for gene transfer vector was necessary. Over the past two years, we have worked with Mario Pende and Paul Kelly (Inserm U845, Centre de Recherche Croissance et Signalisation) towards establishing such a facility. Its missionsThe missions of the core facility are: a) to provide assistance for the investigators on the campus for the design and use of gene transfer vectors derived from lentivirus, retrovirus, adenovirus and adeno-associated virus; b) give access to validated and optimized reagents; c) prepare vectors for research and preclinical studies; d) maintain a data base of vectors available on the site and provide assistance for regulatory filing associated with the laboratory use of Genetically Modified Organisms mandatory declarations to the Commission du Génie Génétique; e) facilitate exchanges with other academic or industrial facilities for the production of clinical grade vectors. The facility has been recognized as an emerging structure in 20098 by the GIS IBiSA (Infrastructures en Biologie Santé et Agronomie ; http://www.ibisa.net/). It is accessible from the IRNEM website since July 2008 ( http://www.necker.fr/irnem/structure/sc/vecteurs_viraux/vecteur_viraux.html)
Literature cited
1. Hacein-Bey-Abina, S., et al., Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Invest, 2008. 118(9): p. 3132-42.
2. Maniatis, T. and R. Reed, An extensive network of coupling among gene expression machines. Nature, 2002. 416(6880): p. 499.
3. Buratti, E., M. Baralle, and F.E. Baralle, Defective splicing, disease and therapy: searching for master checkpoints in exon definition. Nucleic Acids Res, 2006. 34(12): p. 3494-510.
4. Johnson, J.M., et al., Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science, 2003. 302(5653): p. 2141-4.
5. Tazi, J., S. Durand, and P. Jeanteur, The spliceosome: a novel multi-faceted target for therapy. Trends Biochem Sci, 2005. 30(8): p. 469-78.
6. Singh, N.K., et al., Splicing of a critical exon of human Survival Motor Neuron is regulated by a unique silencer element located in the last intron. Mol Cell Biol, 2006. 26(4): p. 1333-46.
7. Skordis, L.A., et al., Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts. Proc Natl Acad Sci U S A, 2003. 100(7): p. 4114-9.
8. Goyenvalle, A., et al., Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping. Science, 2004. 306(5702): p. 1796-9.
9. Quenneville, S.P., et al., Autologous transplantation of muscle precursor cells modified with a lentivirus for muscular dystrophy: human cells and primate models. Mol Ther, 2007. 15(2): p. 431-8.
10. Fraldi, A., et al., Functional correction of CNS lesions in an MPS-IIIA mouse model by intracerebral AAV-mediated delivery of sulfamidase and SUMF1 genes. Hum. Mol. Genet., 2007. 16(22): p. 2693-2702.
