131 Targeted Genome Editing in Spinal Muscular Atrophy Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy S53 Gene TarGeTinG and Gene CorreCTio[.]
Gene Targeting and Gene Correction I (FXN) gene The DNA conformation of the repeat has been reported to cause transcriptional issues as mRNA levels drop significantly in FRDA patients Cells derived from the YG8R mouse model present null mouse FXN genes but contain a FXN transgene obtained from a FRDA human patient We used different pairs of RNA guide combinations and target the pre- and post-GAA sequence in mouse YG8R and human FRDA cells in order to remove completely the repeat expansion Depending of the pair, between 1% and 15% of clones derived from YG8R cells show complete deletion of the GAA repeat The analysis of the YG8R clones revealed some discrepancies between clones obtained from different pairs of transfected RNA guides and further analyses are under investigation The deletion of the GAA repeats expansion might be a highly valuable gene therapy approach for FRDA patients 129 Vector and Cell Mediated BMPR2 Therapy Ameliorates PAH Rebecca L Harper,1 Feng Feng,1 Paul N Reynolds.1 Thoracic Medicine, Royal Adelaide Hospital, Adelaide, South Australia, Australia Inherited pulmonary arterial hypertension is a fatal disease caused by mutations in, and reduced expression of, the bone morphogenetic type receptor (BMPR2), leading to abnormal proliferation of pulmonary vascular cells and obliteration of small pulmonary vessels BMPR2 expression is also reduced in secondary forms of PAH We previously showed that upregulation of BMPR2 expression in the pulmonary vascular endothelium achieved by an endothelialtargeted adenoviral vector ameliorated PAH in rat models In the current work, we evaluated a similar vector approach in a transgenic, BMPR2-mutation related PAH model, further characterised signaling mechanisms involved in the rat model, and extended the approach to the use of ex vivo transduced endothelial progenitor cells (EPCs) as a cell therapy approach to BMPR2 upregulation Methods: SM22-tet / R899x mice were fed doxycycline-containing chow to induce expression of BMPR2 dominant negative protein Mice were then injected via the tail vein with a pulmonary vascular targeted AdBMPR2-conjugate complex (or control vector), and right ventricular pressure measured (RVSP) by transthoracic puncture For the EPC approach, rat bone marrow-derived EPCs were cultured in endothelial selective media then transduced ex-vivo with AdBMPR2 for 48 hours The transduced cells (or untransduced controls) were then injected via tail vein into rats that had induced PAH via subcutaneous monocrotaline injection (60mg/kg) 10 days previously Results: In the murine experiment, BMPR2 delivery led to a 24% reduction of RVSP compared to disease and vector control animals Analysis of whole lung lysate using RT-PCR revealed an increase in eNOS expression in the AdBMPR2 treated group This effect was confirmed in vitro by transduction of cultured endothelial cells, and further confirmed by demonstration of increased NO production The Ad vector approach in the rat MCT model ameliorated PAH as previously described, an effect associated with an increase in Smad1/5/8 phosphorylation and decreased Smad3 phosphorylation In the EPC approach, PAH was also ameliorated following EPC+AdBMPR2 treatment, demonstrated by significant reduction compared to MCT only and EPC only for Fulton Index (26% and 16% respectively), RVSP (34% and 36%) and mPAP (25% and 16%), while analysis of downstream signaling is ongoing Conclusion: These findings indicate that gene delivery strategies via direct vector approaches or cell therapy to upregulate in BMPR2 in the pulmonary vasculature have therapeutic potential in PAH Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy 130 Gene Editing of the AAVS1 Locus with CRISPR/Cas9 Mathieu Vieira,1 Ana Buj Bello,1 Florence G Le Roy.1,2 Genethon, Evry, France; 2University Evry Val-d’Essonne, Evry, France One strategy for gene therapy is gene editing with engineered nucleases such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) or the CRISPR/Cas9 system The latter uses the Cas9 endonuclease and a small guide RNA (sgRNA) to direct specific DNA double-stranded breaks at the chosen target site Once the DNA breaks have occurred, they are repaired by the natural process of homologous recombination (HR) The introduction in the cell of a HR donor template allows replacement of a defective gene with a normal allele at its natural location In order to improve the Cas9 nuclease activity and specificity for a target sequence, we altered the structure of the sgRNA by shortening the sequence complementary to the target site and lengthening the binding sequence of Cas9 We tested the efficacy of these modified sgRNAs on the adeno-associated virus integration site (AAVS1) in human cells transduced by lentiviral vectors We used an Integrationdeficient Lentiviral vector (IDLV) carrying both cassettes of Cas9 and a modified sgRNA targeting AAVS1, together with another IDLV carrying a GFP donor cassette integrating at AAVS1 site after HR We observed similar level of Cas9 nuclease activity with sgRNAs bearing shorter complementary sequence, compared to the non-modified sgRNAs, at the AAVS1 site Moreover, by increasing the length of the scaffold RNA part of the sgRNA, we significantly increased the rate of homologous recombination at the AAVS1 site The improvement of the sgRNA structure tested on the AAVS1 locus can benefit to the targeting of other genome sequences by the CRISPR/Cas9 system 131 Targeted Genome Editing in Spinal Muscular Atrophy Annalisa Lattanzi,1 Matteo Bovolenta,1,2 Samia Martin,1 Vincent Mouly,3 Fulvio Mavilio.1 Genethon, Evry, France; 2Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy; 3Institut de Myologie, GH Pitié-Salpétrière, Paris, France Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the survival-motorneuron (SMN1) telomeric gene Deficiencies in the ubiquitous SMN function affect multiple tissues and organs; however neuronal tissue is primarily sensitive, resulting in α-motor neuron degeneration in the ventral horn of the spinal cord with subsequent neuromuscularjunction dysfunction and proximal muscle weakness The onset of disease and degree of severity are variable in patients and they are determined in part by multiple copies of the centromeric homologue SMN2 that inversely correlate with the phenotypic severity Indeed, SMN2 gene mainly produces a truncated form SMN∆7 by aberrant alternative splicing and a small amount (~10%) of the fully active fulllength SMN, thus buffering the SMN deficiency A potential strategy for treating SMA patients is to increase SMN levels in the affected tissues, hence gene therapy and modifiers of SMN2-alternative splicing have proved therapeutic efficacy in SMA animal models In this study, we explored the possibility of applying targeted genome editing technology to the human SMN locus in order to revert the SMN2 sequence to a SMN1-like sequence that may undergo proper splicing under the the endogenous transcriptional control The resulting correction would be permanent and lead to longlasting protein production in gene-edited cells We used the streptococcus pyogenes Cas9-CRISPR system to target the SMN2 gene at different locations Two main strategies were explored: i) SMN1_exon7 addition/correction by promoting homology-driven DNA repair, ii) SMN2_intron7_ intronic-splicing-silencer (ISS-N1) S53 Gene Targeting and Gene Correction I mutation and correction of SMN2 aberrant splicing, by exploiting the non-homologous end-joining (NHEJ) pathway Plasmids encoding Cas9-GFP under the control of CMV promoter, and selected gRNAs downstream to the Pol-III U6 promoter (Addgene) were transfected in HEK-293T cell line and in immortalized myoblasts derived from either healthy donors or SMA patients Transfection efficiency was estimated as percentage of GFP-expressing cells (20-50% and 1-10%, respectively) and nuclease activity detected by Surveyor assay and target site sequencing In particular, in SMA patient-derived myoblasts we detected mutations (indels) at the level of the induced DNA double-strand break at ~30% frequency Levels of SMN restoration will be investigated by qPCR of the different species of SMN transcripts and by western blotting of SMN protein The goal of this study is to provide an in vitro proof of principle of effective gene correction in SMA patient-derived cells In the context of a multisystemic, complicated disease such as SMA, targeted genome editing strategy could represent an additional therapeutic tool 132 Gene Therapy With Self-Complementary Recombinant Adeno-Associated Virus in Models of Autosomal Dominant Retinitis Pigmentosa Cause by RHO Mutations Brian Rossmiller,1 Danny Zakria,1 Arathi Nandyala,1 Hiral Jivanji,1 Lewin Alfred.1 Molecular Genetics and Microbiology, The University of Florida, Gainesville, FL Purpose Retinitis pigmentosa is the leading hereditary cause of blindness with 30-40% of cases attributable to autosomal dominant retinitis pigmentosa (ADRP) ADRP arises from mutations in at least 24 known genes with 30% arising in the rhodopsin gene (RHO) Given the large heterogeneity of mutations in RHO leading to ADRP, we propose knocking down of endogenous RHO and replacing it with a “hardened” copy, or a RHO with nucleotide changes that preserve the amino acid sequence but decrease the efficiency of knock-down Here we report the use of a scAAV serotype (Y733F) to express a hardened human rhodopsin (hRHO) under the control of the human opsin proximal promoter (HOPS) and an H1 promoter driven shRNA Methods Four different knock-down methods were tested, ribozyme (Rz) 407 and Rz525, miRNA 301 and shRNA 301 against both the wildtype and hardened RHO target regions The transfections were done in HEK293 cells (n=6) with (1) the target plasmid psiCheck2 dual luciferase containing RHO target region, (2) plasmid expressing the shRNA, miRNA or ribozyme against the target region and (3) a control miRNA The reduction in expression of luciferase was measured at 24 and 48 hours post transfection We are using adeno-associated virus vectors to deliver these RNA knockdown agents to mouse models of ADRP: Rho I307N and human RHO transgenic T17M and P23H Mice were injected with AAV-HOP-hRHO and knockdown agents at postnatal day I307N Rho mice were created through the use of N-ethyl-Nnitrosourea (Budzynski et al JBC 2010) The I307N mouse model exhibits very slow degeneration under ambient light but is reduced in visual response to light by 50% in one week post exposure to 10,000 lux Intravitreal injections with one of the two constructs, hRHO+shRNA301, or hRHO+shRNA750 in one eye and AAVHOPS-mCherry or sham injection in the other The mice were followed using electroretinogram and optical coherence tomography Results The knock-down results show shRNA301 and ribozyme 525 to cause the largest reduction of RHO mRNA At one month post injection there was statistically significant difference between T17M RHO eyes injected with AAV-hRHO-shRNA750 and the sham injected eyes S54 Conclusions We have generated a series of combination RNA knockdown and replacement AAV vectors that may be useful for the treatment of ADRP At early time points, our tests of these specific vectors have not been conclusive The injected mice will be followed for longer intervals and additional mice will be added to the study to determine if the difference in visual function of the experimentally treated eyes versus the control is statistically significant 133 Use of 2As To Control Protein Subcellular Localization Ekaterina Minskaia,1 Claire Roulston,1 Garry Luke,1 Martin D Ryan.1 Biomedical Sciences Research Complex (BSRC), University of St Andrews, St Andrews, United Kingdom A substantial proportion (~39%) of all human proteins are either secreted from the cell, located within the lumen/ membranes of cytoplasmic vesicular structures, or, are plasma membrane proteins Given that such high proportion of proteins are initially translocated into the endoplasmic reticulum (ER), many therapeutic strategies rely on the ability to co-express multiple proteins – some, or all of which, might be targeted to such sites This directly applies to in vivo gene therapy strategies, or, when therapeutic proteins may need to be co-expressed with selectable markers (e.g ex vivo gene therapies) As was shown before, Picornavirus 2A (foot-and-mouth disease virus 2A; F2A) and ‘2A-like’ sequences are powerful tools that allow multiple proteins to be translated and co-expressed from a single transcript mRNA under the control of only one promoter When 2A is positioned between sequences encoding two, or more, genes, it mediates a co-translational ‘cleavage’ at its own C-terminus A major problem with co-expression of certain proteins targeted to, or transiting through, the ER is that the ‘cleavage’ activity of short F2As can be greatly inhibited by sequences immediately upstream leading to aberrant sub-cellular localisation of some proteins We have also discovered a number of active cellular 2A-like sequences, associated with non-long terminal repeat (non-LTR) retrotransposons, but also with structural and metabolic proteins: ankyrin repeats, sodium dependent neutral amino acid transporters, and NOD-like receptor (NLR) proteins Examination of the surrounding protein and gene structure revealed that in the majority of cases these 2A sequences occurred as N-terminal features Interestingly, using SignalP, many of these novel 2As scored highly as N-terminal signal peptides Here, we present our latest findings on 2A sequences A series of test proteins (eGFP and mCherry) were expressed i) followed by ‘hybrid’ ‘self-cleaving’ F2A sequences (with different upstream contexts) or ii) downstream of a putative signal 2A We demonstrate that inhibition of F2A-mediated cleavage in shorter sequences can be overcome by introduction of mutations upstream of 2A changing the context of the sequence between the C-terminus of the upstream protein and 2A sequence In the case of N-terminal - cellular - (NLR) 2As, ‘uncleaved’ 2A indeed can act as a signal peptide If 2A does not ‘cleave’, it directs a proportion of the newly synthesised reporter protein to the exocytic pathway: if 2A ‘cleaves’, the protein downstream is localised to the cytoplasm This type of 2A mediates, therefore, a newly discovered form of dual protein targeting Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy ... proteins: ankyrin repeats, sodium dependent neutral amino acid transporters, and NOD-like receptor (NLR) proteins Examination of the surrounding protein and gene structure revealed that in the... N-terminal features Interestingly, using SignalP, many of these novel 2As scored highly as N-terminal signal peptides Here, we present our latest findings on 2A sequences A series of test proteins... SMA, targeted genome editing strategy could represent an additional therapeutic tool 132 Gene Therapy With Self-Complementary Recombinant Adeno-Associated Virus in Models of Autosomal Dominant