B8: Molecular And Cellular Therapeutics

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Flotte et al. (2011) Human Gene Therapy

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used AAV to treat alpha1-antitrypsin deficiency. rAAV-hAAT achieved a peak serum AAT level of 700nm, bu the healthy normal is 40-50uM, and the minimum therapeutic dose is 11uM. Moreover, inflammatory infiltrate (CD3+ and CD8+ cells) was detected in the muscle at the site of injection, and all subjects developed anti-AAV Abs.

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Alain Fischer (2000)

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Gave kids with X-SCID an MLV-based gene therapy. Within 3-4 years, 4 kids developed leukaemia-like disease and 1 had died. This was due to retroviral insertional mutagenesis in the LMO2 oncogene.

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Tolmachova et al. (2013), then MacLaren et al. (2014)

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Successfully transduced AAV2/2.RPE into mouse photoreceptors in vivo and human photoreceptors ex vivo, in the treatment of choroideraemia. They have also shown functional expression in patient fibroblasts. Following on from this pre-clinical work, the first CHM gene therapy was performed in October 2011, and so far 6/6 have had vision improvements (though it is too soon to see if degeneration has been halted)

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Mao et al. (2012)

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Proof of principle for genetic treatment of dominant disease: suppression of mutant allele and delivery of a functioning copy of the gene. Showed in a mouse model (P23H RHO transgenic mouse) of autosomal dominant Retinitis Pigmentosa that a single AAV vector expressing both RHO replacement cDNA and siRNA against P23H RHO mRNA could arrest retinal degeneration 2 months post-injection.

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Yusa et al. (2014)

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Performed a CRISPR screen of the mouse genome to find LOF mutations that altered resistance to C.septicum alpha-toxin or 6-thioguanine (and identified 27 known and 4 new genes). This library is now being used to create mutations in cancer cell lines, to determine which cells are involved in acquiring resistance to treatment.

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Liu et al. (2008)

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- Jx594: oncolytic vaccinia virus that has had several functions deleted: B18R, thus cannot suppress IFN response; Viral TK, thus cannot create phosphorylated T required for DNA replication; VGF, thus cannot induce cell cycle entry. Also induced to express GM-CSF, which has anti-tumour effects. Liu et al. (2008) showed this to have anti-tumour effects in animal models of hepatocellular carcinoma, and phase 1 showed anti-tumour effects in 3/3. Phase 2 highlighted that injections had to be intra-tumour instead of IV, because patients had been vaccinated against smallpox, thus had vaccinia Abs.

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Fire and Mello (1998) Nature

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Were the first to show RNAi in C.elegans. Injected sense and antisense RNA for unc22, a non-essential myofilament protein, that, when deleted, produces a characteristic twitching phenotype and muscle structural defects. When injected separately, little response was seen, and the magnitude of response was much greater when co-injected. Electrophysiological studies revealed that most of the RNA injected had annealed as dsRNA, showing this to be the molecule involved. Also important: injection into the body cavity led to gene expression changes in the gonads, showing a mechanism by which these RNA could be transported from cell to cell: exosomes!

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Harper et al. (2005) PNAS

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Used AAV-mediated RNAi to target expression of the human htt gene in a mouse model of HD. Though phenotypic improvements were seen, they decided to study expression of htt in the cerebellum, which is not related to HD pathology :(

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Valadi et al. (2007) Nat Cell Biol.

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Showed mouse and human mast cell line exosomes to contain RNA (shown by microarray). In vitro translation proved the mRNA to be functional, and total RNA analysis also showed miRNA to be present.

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Wood et al. (2011) Nature Biotech

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Successfully targeted siRNA-containing exosomes to neurons in a mouse. (use the Rabies Virus Glycoprotein to target the exosomes to AchR-containing neurons).

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Ramaswamy et al. (2009)

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Performed stereotactic injection into the striatum of N171-82Q transgenic mouse models of AAV2-NRTN (Neurturn – neurotrophic factor – neuroprotection). This resulted in reduced clasping behaviour and improved stride length, and protected striatal as well as PFC cortical neurons (distant from the injection site).

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McBride et al. (2011)

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Showed in Macaque monkey that injection into the striatum or intravenously of AAV-mtHTT siRNA resulted in a 45% knockdown of the wildtype allele, but that this was well-tolerated and safe, thus RNAi could be used as a therapy for HD.