lumbar up to cervical region showed gene expression. The infected cells were mainly distributed in the posterior funiculus and gray matter. In addition, all 4 ...
Neurologic Diseases (Including Ophthalmic and Auditory Diseases) III lumbar up to cervical region showed gene expression. The infected cells were mainly distributed in the posterior funiculus and gray matter. In addition, all 4 vectors infected dorsal root ganglia neurons very efficiently. Immunostaining showed AAVH43, AAVH48 and AAVHH67 vectors with a CMV promoter infected mainly neurons but not glial cells, similar to AAV9. In addition, AAVH43 and AAVH48 had some common characteristics and infected cerebellar cortex much more efficiently than AAV9. Those neurons showed distinctive morphology as Purkinje cells. Further confirmation with cell-type specific antibodies is needed. Conclusion: The AAVH43, AAVH48 and AAVHH67 vectors have similar ability to infect the spinal cord and dorsal root ganglia, equivalent to AAV9, when delivered by intrathecal injection. Moreover, the cerebellar cortex is more susceptible to AAVH43 and AAVH48 infection. With further investigation, the three novel AAV serotypes could be suitable alternative candidates for CNS gene delivery and therapy.
604. TALE-VP64 Targeting the Frataxin Promoter Increase the Expression of That Gene in Friedreich Fibroblasts Jacques P. Tremblay,1 Joël Rousseau,1 Pierre Chapdelaine.1 1 Médecine Moléculaire, Université Laval, Québec, QC, Canada.
Friedreich’s ataxia (FRDA) is due to a reduced frataxin expression due to a trinucleotide repeat. It is 30% in patients with 200 GAA repeats and only 5% in patients with 900 GAA repeats 33. However, carriers of this disease produce about only 50% of the normal level of frataxin but do not develop symptoms. We have engineered 12 genes coding for TALE proteins targeting different nucleotide sequences present in the frataxin promoter. Each expression plasmid contains a TALEFrat gene fused with a transcription activator, VP64 under the EF1α promoter, a 2A peptide and an EGFP. When one of these plasmids was transfected alone in human cells, only green fluorescence was detected by FACS indirectly confirming the expression of the TALEFrat/VP64 protein. To identify which of our 12 different TALEFrat/VP64 proteins were able to better induce the expression of the frataxin gene, we have constructed a reporter plasmid containing the proximal region of the frataxin promoter, followed by a minimal CMV promoter and a mCherry reporter gene (pCR3.1 proximal-promoter-frataxin-miniCMV-mCherry). This reporter plasmid was initially transfected in human cells alone. Very few cells expressed the red fluorescence because the promoter was not effective without transactivation by binding factors. When the reporter plasmid was co-transfected in human cells with one of the pCR3.1-TALEFrat/VP64-2A-EGFP plasmids, a much higher number of cells expressed the red fluorescence because the TALEFrat/ VP64 attached to the proximal frataxin promoter and induced the transcription of mCherry. The 3 TALEFrat/VP64, which induced the strongest expression of mCherry, were targeting promoter sequences close to each other. A plasmid coding for TALEFrat#8/VP64 was nucleofected in normal fibroblasts. Using quantitative RT-PCR, we have confirmed in 3 independent experiments that the expression of the frataxin mRNA (relative to GAPDH mRNA) in human cells was doubled or triple by TALEFrat#8/VP64 when results were normalized with cells transfected with EGFP or non-transfected cells. We have also shown that this TALE also increased by 2 folds the frataxin protein in fibroblasts from a FRDA patient. In a recent preliminary result, we have shown that the transfection of TALEFrat#8/VP64 plasmid in YG8R fibroblasts also increases frataxin mRNA (by about 1.4 to 1.9 fold) and protein (by about 1.4 fold). Such increases would be in the therapeutic range (i.e., 50% of normal frataxin level) for many patients. However, for patients that have less than 25% of the normal level of frataxin expression, a further increase of frataxin would be S240
required and this project may permit to obtain higher frataxin increases and thus this would lead to an increased number of FRDA patients who would benefit from our therapy.
605. Residual Cesium Chloride in AAV Vectors Purified by CsCl Gradient Centrifugation Does Not Cause Obvious Inflammation or Retinal Degeneration in C57Bl6/J Mice Following Subretinal Injection
Karen Guerin,1 Vivian Choi,1 Jorge Aranda,1 John Demirs,1 Hui Li,1 Junzheng Yang,1 Niem Nguyen,1 Steve Bottega,1 Bruce Jaffee,1 Ted Dryja,1 Seshidhar Police.1 1 Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA. Cesium chloride density gradient ultracentrifugation is the conventional method of purification for adeno-associated viral vectors. This method is often preferred because it allows for good separation of full and empty vector particles, and because it can be adjusted to purify any AAV serotype with minimal optimization. AAV vectors purified by CsCl gradient centrifugations have been evaluated in many preclinical studies both in small and large animals, and in clinical studies for ophthalmology disease indications. Most CsCl is removed at the end of the purification process when buffer exchange is done by dialysis, but the level of residual CsCl has not been reported. Furthermore, there has been little or no evaluation of the potential toxicity of residual CsCl in the final AAV product. The purpose of this work was to measure the amount of residual CsCl in purified AAV vectors and investigate its potential toxicity following subretinal injection in C57Bl6/J mice.CsCl in 20 purified AAV vectors was measured by inductively coupled plasma mass spectrometry. The amount of CsCl delivered with a subretinal injection of 1x109 vector genomes was calculated for each vector based on a 1-µl injection volume and the vector’s titer. CsCl levels were found to be in the range of 0.3 ng to 5 ng per vector dose. To determine whether CsCl is toxic at this level, we performed a dose-response study in which our formulation buffer was spiked with known amounts of CsCl, ranging from 0-100 ng per injection. We used fundus imaging and microglial cell counts to evaluate inflammatory responses and H&E staining of paraffin-embedded eyes for histopathology assessment 6 weeks post injection. The lowest level of residual CsCl measured in our AAV vector preps was on the order of 0.3 ng in a dose of 1x109vg. Extended buffer exchanges did not result in complete removal of CsCl from the vector preparations, suggesting that CsCl may be associated with the AAV particles. A dose-response study with CsClpurified AAV vectors was carried out to determine whether residual CsCl in the presence of AAV could be detrimental. Null AAV2 and AAV8 vectors were tested at three different doses (1x108 vg, 5x108 vg, 1x109 vg/eye). No evidence of inflammation or damage to the retina or RPE could be seen on the fundus images. These observations were confirmed by the histopathology assessment and a count of the number of microglial cells in the retina and posterior eyecup. We did not see evidence of inflammation, even at the highest dose of 100 ng of CsCl. On H&E-stained sections retinal thinning was seen only at the inferred injection sites, and this observation was consistent across all dose groups. Finally we tested a null vector purified by affinity chromatography, a CsCl-free process. As in our previous studies we found no evidence of inflammation or retinal damage. In conclusion we did not find evidence that residual CsCl from an AAV purification process is toxic to the retina following subretinal injection.
Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
