1019. Development of Tissue-Specific RNAi for Gene Therapy

OLIGONUCLEOTIDE THERAPIES B 1018. Stem Cell Gene Therapy: Toward a Universal Platform Relying on Custom Endonuclease-Boosted Gene Targeting, Cybridization, Transient Regenerative/Epigenetic Gene Therapy and Synergistic Combinations Roger Bertolotti. 1 CNRS, Gene Therapy and Regulation Research, Faculty of Medicine, University of Nice-Sophia Antipolis, Nice, France. A platform has been devised in which autologous stem cells are the targets of both long-term and transient gene therapy culminating in synergistic combinations. Gene targeting and cybridization drive long-term gene therapy: they mediate gene repair/alteration or targeted transgene integration, and mitochondrial (mt)DNA transfer, respectively. Transient regenerative gene therapy drives regenerative medicine sensu stricto (aging/degenerative pathologies) and synergizes long-term gene therapy, thereby magnifying the repopulating/ regenerative ability of target stem cells/cybrids (inherited/acquired disorders including mtDNA diseases). Transient gene therapy has an epigenetic arm for long-term gene inactivation mediated by promoter-specific siRNAs (Morris et al, 2004); conversely, gene activation might possibly be amenable to non-coding antisense RNAs. Such a universal platform is aimed at eliminating hazardous randomintegration of therapeutic DNA, at reversing inherited diseases by reestablishing wild-type genomic homeostasis and at tackling most pathologies through stem cell repopulation dynamics into appropriate niches (long-term engraftment) and tissues (cell turnover). Conventional gene targeting is overwhelmed by random integration and is inefficient unless a double-strand break (DSB) hits target chromosomal DNA. Emerging DSB-boosted gene targeting relies on chimeric zinc-finger endonucleases (ZFNs) that create site-specific DSBs. Optimization of zinc-finger DNA-binding domains culminates in targeting efficiencies compatible with clinical single-base correction: homologous recombination mediates strand exchanges between chromosomal and transfecting therapeutic DNA under negligible random integration of both therapeutic DNA and ZFN vectors (Urnov et al, 2005). However, either with ZFNs or emerging custom homing endonucleases, sequence modification efficiency is restricted to a ∼50 bp distance from the DSB, thereby allowing short multi-base mutation handling but preventing clinical management of long deletions/insertions and custom site-specific integrative gene therapy. Thus, in order to reach full clinical potentialities, vectorisation of therapeutic DNA might need optimization based on our recombinaseDNA nucleoprotein complexes/DNA backbones or on viral gene targeting. An alternate approach is related to our platform mtDNA arm that substitutes stem cell cybrids for current allotopic transgene expression: stem cells are cured ex vivo from endogenous mitochrondria and repopulated by wild-type mitochondria upon fusion with enucleated cells (cybridization). This cybrid regenerative therapy includes a selective rescue in vitro and is thus tailored for stem cells with an extensive in vitro growth potential. Such stem cells are thus amenable to in vitro post-gene targeting selection, thereby enabling efficient exchange of sizeable DNA sequences culminating in clinical targeted transgene integration. Our platform is thus discussed in terms of improved endonuclease-boosted gene targeting, somatic stem cell amplification/cybridization, transient regenerative/epigenetic gene therapy and synergistic associations.

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OLIGONUCLEOTIDE THERAPIES B 1019. Development of Tissue-Specific RNAi for Gene Therapy Danny Allen,1 Paul F. Kenna,1 Arpad Palfi,1 Helena McMahon,1 Sophia Millington-Ward,1 Mary O’Reilly,1 Peter Humphries,1 G. Jane Farrar.1 1 Department of Genetics, Trinity College Dublin, Dublin, Ireland. RNA interference (RNAi) is a post-transcriptional, sequencespecific gene-silencing technology that utilises double stranded (dsRNA) molecules to degrade messenger RNA containing the same sequence as the dsRNA. Although a significant body of research has been undertaken in the field of suppression (utilising RNAi) in mammalian cells and animals, in the majority of these studies synthetic siRNAs or else polymerase III promoter-driven shRNAs have been utilised. Notably, neither of these methods allow for temporal or spatial control of suppression. Whilst there are some studies describing methods of conditional RNAi (e.g. using cre recombinase), the current study represents the first demonstration of a single plasmid with components that enable tissue specific suppression in vitro and in vivo. In essence, the approach combines tissue specific polymerase II promoters with cis-acting hammerhead ribozymes and short-hairpin RNA (shRNA) sequences to generate potent dsRNA molecules in tissues defined by the promoter in use. Results in cell culture and in mice demonstrating that the approach outlined above can be utilized to provide tissue specific RNAi will be presented. More specifically, potent suppression of EGFP expression has been demonstrated in mouse kidney and liver using a cmv promoter driven shRNA construct, whilst an albumin promoter driven shRNA construct has been shown to elicit suppression only in mouse liver. Tissue-specific RNAi has several advantages over traditional methods not least of which is the ability to minimise offtarget effects of RNAi. Additionally, tissue-specific RNAi, in principle, greatly increases the resolution of RNAi technology facilitating the ability to silence genes in individual tissues which, if down-regulated in all tissue types, may be lethal.

1020. AAV Delivery of Suppression and Replacement Constructs for Rhodopsin-Linked Autosomal Dominant Retinitis Pigmentosa Naomi Chadderton,1 Arpad Palfi,1 Mary O’Reilly,1 Sophia Millington-Ward,1 Marius Ader,1 Markus Hildinger,2 Alberto Auricchio,2 Gearoid Tuohy,1 Peter Humphries,1 Paul F. Kenna,1 G. Jane Farrar.1 1 Genetics, Trinity College Dublin, Dublin, Ireland; 2Department of Pediatrics, TIGEM, Napoli, Italy. Retinitis pigmentosa (RP) is a genetically heterogeneous disorder that can be inherited in an autosomal dominant, autosomal recessive, x-linked, digenic or mitochondrial fashion. One form of autosomal dominantly inherited RP (adRP) is due to mutations in the rhodopsin gene (RHO) which encodes the light sensitive pigment rhodopsin, the first component in the visual transduction cascade in rod photoreceptor cells. Over one hundred mutations have been characterised in this gene to date. Given the immense mutational heterogeneity present in RHO-linked RP, a therapeutic strategy termed suppression and replacement focused on correcting the primary genetic defect while circumventing the mutational heterogeneity present in rhodopsin-linked adRP has been proposed. The approach involves suppression of mutant and wild type RHO mRNA, together with provision of a replacement gene, which is

Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy