- Email: [email protected]
125. AAV-Mediated Gene Correction for the Treatment of Recessive Dystrophic Epidermolysis Bullosa
AAV GENOME & HOST INTERACTIONS 123. Absence of Integration Hotspots in Mice after Intramuscular Injection of AAV1-LPLS447X
C. Kaeppel,1 S. Beattie,2 A. Nowrouzi,1 U. Appelt,1 R. Kirsten,1 H. Glimm,1 J. Snapper,2 C. von Kalle,1 H. Petry,2 M. Schmidt.1 1 German Cancer Research Center (DKFZ) and National Center for Tumor Diseases (NCT), Heidelberg, Germany; 2Amsterdam Molecular Therapeutics (AMT), Amsterdam, Netherlands. Since recombinant AAV vectors are considered to integrate into host chromosomes at a very low frequency (<1%), we sought to analyse the persistence and integration frequency of AAV1-LPLS447X in mice after intramuscular injection. In the first study, 1E13 gc/kg were injected intramuscularly into 4 or 13 week old mice and adductors from the hind limb were collected 4 weeks post-injection. In a second approach, 7-8 week old mice received 6E13 gc/kg intramuscularly and hind limb muscles and livers were collected 4 or 12 weeks post-injection. LAM-PCR was performed on the 3-prime end of the vector using the two different restriction endonucleases MseI and Tsp509I. >16000 (study 1) and >25000 (study 2) AAV derived LAM-amplicons were 454 pyrosequenced, revealing 401 and 856 unique integration sites (IS), respectively. The chromosomal distribution and the distribution in respect to gene coding regions showed that the integration of the AAV vector is random in the host genome. Evaluation of integration hotspots by common integration sites (CIS) analysis revealed no CIS of higher order than 2nd in study 1 and no CIS of higher order than 6th in study 2 indicating the absence of clonal skewing. Furthermore, the calculated relative sequence count of each unique IS was low, indicating the absence of dominating clones. Despite a high number of IS being detected, the presence of a high frequency of concatemers (up to 97%) was demonstrated in each sample. The feasibility of complete inverted terminal repeat (ITR) sequencing could be shown (0,018% of all sequences in study 1). As expected, partially deleted ITRs with preferred breakpoints within the first half of the ITR (starting from the sequencing initiating site) were detected in our settings. Taken together, these findings are unique in their context showing random AAV vector integration and are of clinical relevance for ongoing and upcoming clinical trials.
124. Adeno-Associated Viral Vector DNA Is Insensitive to De Novo CpG Methylation but Rapidly Associates with Histone Modifications in Liver and Skeletal Muscle
Adrien Léger,1 Caroline Le Guiner,1,2 Michael L. Nickerson,3 Nicolas Ferry,4 Philippe Moullier,1,2,5 Richard O. Snyder,1,5,6 Magalie Penaud-Budloo.1,5 1 INSERM UMR649, Nantes, France; 2Genethon, Evry, France; 3 Cancer and Inflammation Program, National Cancer Institute - NIH, Frederick, MD; 4INSERM UMR948, Nantes, France; 5 Department of Molecular Genetics and Microbiology, University of Florida, College of Medecine, Gainesville, FL; 6Center of Excellence for Regenerative Health Biotechnology, University of Florida, Alachua, FL. Recombinant Adeno-Associated Virus (rAAV) vector-mediated gene transfer allows long-term transgene expression in quiescent tissues. We previously showed in nonhuman primate (NHP) skeletal muscle that rAAV genomes are assembled in nucleosomes and form a stable chromatin structure. In the mammalian nucleus, chromatin remodeling via epigenetic modifications plays a key role in transcriptional regulation. In order to assess the impact of such regulation on the expression of a therapeutic protein, we injected mice and NHP via the intramuscular (IM) or the intravenous (IV) route with a rAAV2/1 or rAAV2/8 vector carrying a reporter transgene under the control of the constitutive Rous Sarcoma Virus promoter (RSVp). To correlate the transcriptional activity with the epigenetic regulation, we determined the vector copy number and the transgene mRNA level Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
from skeletal muscle and liver samples at early and late time-points by quantitative PCR and RT-PCR respectively. DNA methylation analysis was carried out by pyrosequencing after sodium bisulfite treatment and histone post-transcriptional modifications (PTM) associated with the rAAV chromatin were analyzed by chromatin immunoprecipitation (ChIP). No significant CpG methylation was found along the RSVp sequence of rAAV genomes either in the virions or following transduction of murine and nonhuman primate tissues. This finding is consistent with the long-term expression of the transgene and suggests a resilience of rAAV episomes to DNA methylation. Furthermore, we demonstrated that the RSV promoter drives a much higher level of transgene transcription in skeletal muscle than in the liver. As direct de novo DNA methylation is not responsible for this tissue-differential/specific expression, we looked for evidence of other regulatory mechanisms. Indeed, our preliminary ChIP experiments showed that some rAAV genomes are rapidly marked with either repressive or active histone PTM after vector administration.
125. AAV-Mediated Gene Correction for the Treatment of Recessive Dystrophic Epidermolysis Bullosa
Rowan Flynn,1 Lisa M. Petek,1 Andrew P. South,2 Daniel G. Miller.1 1 Department of Pediatrics, University of Washington, Seattle, WA; 2Centre for Oncology and Molecular Medicine, University of Dundee, Dundee, United Kingdom.
Epidermolysis Bullosa (EB) encompasses a group of inherited skin diseases characterized by severe trauma-induced blistering, skin denudation, and in the case of dystrophic EB, an increased risk of squamous cell carcinoma. Dystrophic EB is caused by dominant or recessive mutations in the gene encoding for collagen VII, COL7A1. Collagen VII is a major structural component of anchoring fibrils and is secreted by both keratinocytes and fibroblasts. Recessive dystrophic EB (RDEB) is an excellent candidate for gene therapy due to easy target tissue accessibility. Keratinocytes can be isolated from patient biopsies and transduced ex vivo, circumventing a potential immune response to the delivery vector and improving efficiency. Skin equivalents derived from these cells can then be transplanted back to the patient. Our lab has previously demonstrated the efficacy of AAV-mediated gene targeting as a treatment approach for the dominantly inherited simplex form of epidermolysis bullosa. Transcription of a mutated gene encoding dominantly active keratin was disrupted using an AAV gene knock-out strategy. Here, we demonstrate for the first time, that this technique can also be applied to gene correction by targeting COL7A1 mutations that cause RDEB. Gene correction would offer several advantages over gene addition; large coding regions make it difficult to produce high titer retrovirus vector preparations and the dominantly inherited form cannot be effectively treated with addition strategies. However, random vector integration events associated with AAV can make it difficult to distinguish successfully targeted clones from vector integration at random genomic sites. To address this issue, we designed a vector containing a synthetic exon promoter trap (SEPT) targeting an intron close to the site of an RDEB-causing mutation, Q251X. This promoterless cassette utilizes the COL7A1 promoter for reporter gene expression, preventing detection of random integrants. Attempts at using a SEPT containing an internal ribosomal entry site (IRES) and GFP reporter resulted in weak fluorescence and reduced our ability to detect homologous recombination. We overcame this by using a brighter reporter, DSRed, and replacing the IRES with a 2A sequence. Southern blot confirmed presence of the of this expression cassette in the target intron. Inclusion of loxP sites in our cassette facilitates Cremediated removal of the SEPT, leaving behind corrected COL7A1. By specifically targeting pre-sorted keratinocyte stem cells for S49
AAV GENOME & HOST INTERACTIONS transduction, it should be possible to provide corrected cells of high growth potential suitable for therapeutic use. This is the first study where AAV-targeting has been used for the purpose of gene correction in EB and it demonstrates a technique that can be applied to other disease-causing mutations.
126. A Distinctive Fate of rAAV Vector Genomes in the Liver Transduced with Very Low Doses of rAAV8 in Mice Kei Adachi,1 Hiroyuki Nakai.1 1 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA.
Recombinant AAV (rAAV) vectors mediate persistent transgene expression in vivo primarily from extrachromosomal vector genomes. Although rAAV vectors are usually viewed as non-integrating vectors, we and others have demonstrated that rAAV genomes could integrate into the cellular genomes of somatic cells in mice, including hepatocytes in the liver. The liver is one of the major target organs for therapeutic gene delivery and is often inadvertently transduced with vector particles leaked from remote tissues. On the other hand, rAAV vector integration in the liver has raised a concern due to the demonstration of potential genotoxicity leading to hepatocarcinogenesis in a mouse study. Therefore, the elucidation of the rAAV vector genome fate in the liver is crucial for understanding the vector biology in gene therapy and assessing the risk of potential genotoxicity. Here we report our experimental results indicating that, when rAAV serotype 8 (rAAV8) vectors are intravenously injected into adult mice at doses substantially lower than the standard doses (i.e., 1.0 x 10e7 -1.0 x 10e9 vector genomes (vg) per mouse as opposed to the standard doses of 10e10 to 10e12 vg/mouse), rAAV vector genomes more preferable integrate into chromosomes rather than remaining as non-integrated forms. In our study, we established a very sensitive quantitative PCR (qPCR) assay system by which we could quantitatively and accurately assess rAAV vector genome copy numbers in the liver. The assay system we employed uses 5 micrograms of genomic DNA as qPCR templates, which showed linearity over a 5-log dynamic range down to 0.000034 double-stranded vector genome (ds-vg) per diploid genomic equivalent (ds-vg/dge). Adult C57BL/6 mice were injected via the tail vein with a rAAV8 vector at doses ranging from 5.0 x 10e10 down to 1.0 x 10e7 vg/mouse, and relative vector genome integration efficiencies were indirectly assessed by comparing vector genome copy numbers before and after two-thirds partial hepatectomy (PHx) using Southern blot analysis (when applicable) and the qPCR assay. In wild type mice injected with 5.0 x 10e10 vg/mouse, PHx resulted in 80-85% loss of vector genomes in the liver, demonstrating primarily the extrachromosomal nature of rAAV genomes in the liver. The substantial decrease of copy numbers in this range has been reproducibly demonstrated by us and others in the previous experiments. In contrast and unexpectedly, we found that the effect of PHx on the decrease in vector genome copy numbers was significantly blunted when the mice were injected at doses of 1.0 x 10e9 vg/mouse or lower. For example, in the mice injected with rAAV8 at a dose of 1.0 x 10e8 vg/mouse (n=4), vector genome copy numbers before and after PHx were 0.0013+/-0.0001 and 0.0008+/0.0001 (mean+/-SEM) ds-vg/dge, respectively (38+/-11% decrease). Although this represents indirect evidence, these results indicate that a lesser viral genome load in cells causes activation of cellular DNA repair machinery that mediates genomic integration more preferably than intra- and/or inter-molecular viral genome recombination. In addition, this observation may have implication in the risk assessment of vector spillover into the liver.
S50
127. AAV-Mediated shRNA Integration into an Endogenous miRNA Locus as a New Strategy for Safe, Specific and Persistent Therapeutic RNAi
Stefan Mockenhaupt,1 Dirk Grimm.1 1 Infectious Diseases, Heidelberg University, Cluster of Excellence CellNetworks, Heidelberg, Baden-Wuerttemberg, Germany. The use of viral vector-delivered small hairpin RNAs (shRNAs) to induce RNAi-mediated gene silencing has started to revolutionize human gene therapy due to its efficiency, specificity and versatility. Yet, a critical prerequisite towards clinical translation remains the implementation of potent and safe means to prevent loss of the shRNA vector in proliferating infected cells. Ideally, this will be achieved via stable integration of the shRNA vector DNA into the human genome at a position tolerating insertions while providing sustained and regulated RNAi expression. Here, we accordingly propose sitespecific shRNA integration into an endogenous mi(cro)RNA locus as a novel strategy. For proof-of-concept, we engineered a plasmid to carry the authentic human miR-122 stem-loop structure along with 2.5 or 2 kb of flanking 5’ or 3’ genomic sequences, respectively. We next inserted a potent shRNA targeting human-alpha-1-antitrypsin (hAAT, a liver-borne enzyme) whose stem-loop mimics the miR122 structure into a position located about 100 bp upstream of miR-122 itself. Our hope and rationale were that following eventual integration into the genomic miR-122 locus, the endogenous miR122 promoter will permanently and efficiently co-transcribe the shRNA in a tissue-restricted fashion. Therefore, we first had to validate correct expression and functionality of both hairpin RNAs - ectopic shRNA and genuine miR-122 - encoded by our bicistronic sequence. We thus cloned the entire fragment under a strong viral RNA polymerase II promoter and measured knockdown of luciferase reporters tagged with binding sites for either small RNA. Indeed, we found that both target sequences were efficiently and specifically silenced upon expression of our chimeric plasmid, comparable to extents observed with individual sh or miRNA-encoding constructs. Encouraged by this, we have now transferred the entire fragment into an Adeno-associated viral (AAV) vector context and pseudotyped it with a new synthetic AAV capsid that we have recently engineered to mediate highly potent transduction of cultured cells. At present, we are using this vector in homologous recombination-based attempts to site-specifically integrate our chimeric sh/miRNA sequence into the endogenous miR-122 locus in a human liver cell line. Resulting clones will next be thoroughly characterized and scored positive based on a set of critical parameters, in particular unperturbed cell viability and correct concurrent sh/miRNA expression. Concomitantly, we will assess the potency and longevity of hAAT target gene silencing upon extended culture of single cellular clones. If successful, our approach opens up a wealth of novel avenues to safely integrate ectopic therapeutic shRNAs into human cells and to guarantee tissuespecific and persistent RNAi. We predict that by using tailored and potent viral capsids, and by targeting selected cell-specific miRNA loci, it will be feasible to expand our strategy to numerous clinically relevant organs and to apply the entire approach in vivo. Moreover, while we currently co-express a single shRNA, our construct can be readily modified to harbor multiple hairpins to permit combinatorial RNAi therapies.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
C. Kaeppel,1 S. Beattie,2 A. Nowrouzi,1 U. Appelt,1 R. Kirsten,1 H. Glimm,1 J. Snapper,2 C. von Kalle,1 H. Petry,2 M. Schmidt.1 1 German Cancer Research Center (DKFZ) and National Center for Tumor Diseases (NCT), Heidelberg, Germany; 2Amsterdam Molecular Therapeutics (AMT), Amsterdam, Netherlands. Since recombinant AAV vectors are considered to integrate into host chromosomes at a very low frequency (<1%), we sought to analyse the persistence and integration frequency of AAV1-LPLS447X in mice after intramuscular injection. In the first study, 1E13 gc/kg were injected intramuscularly into 4 or 13 week old mice and adductors from the hind limb were collected 4 weeks post-injection. In a second approach, 7-8 week old mice received 6E13 gc/kg intramuscularly and hind limb muscles and livers were collected 4 or 12 weeks post-injection. LAM-PCR was performed on the 3-prime end of the vector using the two different restriction endonucleases MseI and Tsp509I. >16000 (study 1) and >25000 (study 2) AAV derived LAM-amplicons were 454 pyrosequenced, revealing 401 and 856 unique integration sites (IS), respectively. The chromosomal distribution and the distribution in respect to gene coding regions showed that the integration of the AAV vector is random in the host genome. Evaluation of integration hotspots by common integration sites (CIS) analysis revealed no CIS of higher order than 2nd in study 1 and no CIS of higher order than 6th in study 2 indicating the absence of clonal skewing. Furthermore, the calculated relative sequence count of each unique IS was low, indicating the absence of dominating clones. Despite a high number of IS being detected, the presence of a high frequency of concatemers (up to 97%) was demonstrated in each sample. The feasibility of complete inverted terminal repeat (ITR) sequencing could be shown (0,018% of all sequences in study 1). As expected, partially deleted ITRs with preferred breakpoints within the first half of the ITR (starting from the sequencing initiating site) were detected in our settings. Taken together, these findings are unique in their context showing random AAV vector integration and are of clinical relevance for ongoing and upcoming clinical trials.
124. Adeno-Associated Viral Vector DNA Is Insensitive to De Novo CpG Methylation but Rapidly Associates with Histone Modifications in Liver and Skeletal Muscle
Adrien Léger,1 Caroline Le Guiner,1,2 Michael L. Nickerson,3 Nicolas Ferry,4 Philippe Moullier,1,2,5 Richard O. Snyder,1,5,6 Magalie Penaud-Budloo.1,5 1 INSERM UMR649, Nantes, France; 2Genethon, Evry, France; 3 Cancer and Inflammation Program, National Cancer Institute - NIH, Frederick, MD; 4INSERM UMR948, Nantes, France; 5 Department of Molecular Genetics and Microbiology, University of Florida, College of Medecine, Gainesville, FL; 6Center of Excellence for Regenerative Health Biotechnology, University of Florida, Alachua, FL. Recombinant Adeno-Associated Virus (rAAV) vector-mediated gene transfer allows long-term transgene expression in quiescent tissues. We previously showed in nonhuman primate (NHP) skeletal muscle that rAAV genomes are assembled in nucleosomes and form a stable chromatin structure. In the mammalian nucleus, chromatin remodeling via epigenetic modifications plays a key role in transcriptional regulation. In order to assess the impact of such regulation on the expression of a therapeutic protein, we injected mice and NHP via the intramuscular (IM) or the intravenous (IV) route with a rAAV2/1 or rAAV2/8 vector carrying a reporter transgene under the control of the constitutive Rous Sarcoma Virus promoter (RSVp). To correlate the transcriptional activity with the epigenetic regulation, we determined the vector copy number and the transgene mRNA level Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
from skeletal muscle and liver samples at early and late time-points by quantitative PCR and RT-PCR respectively. DNA methylation analysis was carried out by pyrosequencing after sodium bisulfite treatment and histone post-transcriptional modifications (PTM) associated with the rAAV chromatin were analyzed by chromatin immunoprecipitation (ChIP). No significant CpG methylation was found along the RSVp sequence of rAAV genomes either in the virions or following transduction of murine and nonhuman primate tissues. This finding is consistent with the long-term expression of the transgene and suggests a resilience of rAAV episomes to DNA methylation. Furthermore, we demonstrated that the RSV promoter drives a much higher level of transgene transcription in skeletal muscle than in the liver. As direct de novo DNA methylation is not responsible for this tissue-differential/specific expression, we looked for evidence of other regulatory mechanisms. Indeed, our preliminary ChIP experiments showed that some rAAV genomes are rapidly marked with either repressive or active histone PTM after vector administration.
125. AAV-Mediated Gene Correction for the Treatment of Recessive Dystrophic Epidermolysis Bullosa
Rowan Flynn,1 Lisa M. Petek,1 Andrew P. South,2 Daniel G. Miller.1 1 Department of Pediatrics, University of Washington, Seattle, WA; 2Centre for Oncology and Molecular Medicine, University of Dundee, Dundee, United Kingdom.
Epidermolysis Bullosa (EB) encompasses a group of inherited skin diseases characterized by severe trauma-induced blistering, skin denudation, and in the case of dystrophic EB, an increased risk of squamous cell carcinoma. Dystrophic EB is caused by dominant or recessive mutations in the gene encoding for collagen VII, COL7A1. Collagen VII is a major structural component of anchoring fibrils and is secreted by both keratinocytes and fibroblasts. Recessive dystrophic EB (RDEB) is an excellent candidate for gene therapy due to easy target tissue accessibility. Keratinocytes can be isolated from patient biopsies and transduced ex vivo, circumventing a potential immune response to the delivery vector and improving efficiency. Skin equivalents derived from these cells can then be transplanted back to the patient. Our lab has previously demonstrated the efficacy of AAV-mediated gene targeting as a treatment approach for the dominantly inherited simplex form of epidermolysis bullosa. Transcription of a mutated gene encoding dominantly active keratin was disrupted using an AAV gene knock-out strategy. Here, we demonstrate for the first time, that this technique can also be applied to gene correction by targeting COL7A1 mutations that cause RDEB. Gene correction would offer several advantages over gene addition; large coding regions make it difficult to produce high titer retrovirus vector preparations and the dominantly inherited form cannot be effectively treated with addition strategies. However, random vector integration events associated with AAV can make it difficult to distinguish successfully targeted clones from vector integration at random genomic sites. To address this issue, we designed a vector containing a synthetic exon promoter trap (SEPT) targeting an intron close to the site of an RDEB-causing mutation, Q251X. This promoterless cassette utilizes the COL7A1 promoter for reporter gene expression, preventing detection of random integrants. Attempts at using a SEPT containing an internal ribosomal entry site (IRES) and GFP reporter resulted in weak fluorescence and reduced our ability to detect homologous recombination. We overcame this by using a brighter reporter, DSRed, and replacing the IRES with a 2A sequence. Southern blot confirmed presence of the of this expression cassette in the target intron. Inclusion of loxP sites in our cassette facilitates Cremediated removal of the SEPT, leaving behind corrected COL7A1. By specifically targeting pre-sorted keratinocyte stem cells for S49
AAV GENOME & HOST INTERACTIONS transduction, it should be possible to provide corrected cells of high growth potential suitable for therapeutic use. This is the first study where AAV-targeting has been used for the purpose of gene correction in EB and it demonstrates a technique that can be applied to other disease-causing mutations.
126. A Distinctive Fate of rAAV Vector Genomes in the Liver Transduced with Very Low Doses of rAAV8 in Mice Kei Adachi,1 Hiroyuki Nakai.1 1 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA.
Recombinant AAV (rAAV) vectors mediate persistent transgene expression in vivo primarily from extrachromosomal vector genomes. Although rAAV vectors are usually viewed as non-integrating vectors, we and others have demonstrated that rAAV genomes could integrate into the cellular genomes of somatic cells in mice, including hepatocytes in the liver. The liver is one of the major target organs for therapeutic gene delivery and is often inadvertently transduced with vector particles leaked from remote tissues. On the other hand, rAAV vector integration in the liver has raised a concern due to the demonstration of potential genotoxicity leading to hepatocarcinogenesis in a mouse study. Therefore, the elucidation of the rAAV vector genome fate in the liver is crucial for understanding the vector biology in gene therapy and assessing the risk of potential genotoxicity. Here we report our experimental results indicating that, when rAAV serotype 8 (rAAV8) vectors are intravenously injected into adult mice at doses substantially lower than the standard doses (i.e., 1.0 x 10e7 -1.0 x 10e9 vector genomes (vg) per mouse as opposed to the standard doses of 10e10 to 10e12 vg/mouse), rAAV vector genomes more preferable integrate into chromosomes rather than remaining as non-integrated forms. In our study, we established a very sensitive quantitative PCR (qPCR) assay system by which we could quantitatively and accurately assess rAAV vector genome copy numbers in the liver. The assay system we employed uses 5 micrograms of genomic DNA as qPCR templates, which showed linearity over a 5-log dynamic range down to 0.000034 double-stranded vector genome (ds-vg) per diploid genomic equivalent (ds-vg/dge). Adult C57BL/6 mice were injected via the tail vein with a rAAV8 vector at doses ranging from 5.0 x 10e10 down to 1.0 x 10e7 vg/mouse, and relative vector genome integration efficiencies were indirectly assessed by comparing vector genome copy numbers before and after two-thirds partial hepatectomy (PHx) using Southern blot analysis (when applicable) and the qPCR assay. In wild type mice injected with 5.0 x 10e10 vg/mouse, PHx resulted in 80-85% loss of vector genomes in the liver, demonstrating primarily the extrachromosomal nature of rAAV genomes in the liver. The substantial decrease of copy numbers in this range has been reproducibly demonstrated by us and others in the previous experiments. In contrast and unexpectedly, we found that the effect of PHx on the decrease in vector genome copy numbers was significantly blunted when the mice were injected at doses of 1.0 x 10e9 vg/mouse or lower. For example, in the mice injected with rAAV8 at a dose of 1.0 x 10e8 vg/mouse (n=4), vector genome copy numbers before and after PHx were 0.0013+/-0.0001 and 0.0008+/0.0001 (mean+/-SEM) ds-vg/dge, respectively (38+/-11% decrease). Although this represents indirect evidence, these results indicate that a lesser viral genome load in cells causes activation of cellular DNA repair machinery that mediates genomic integration more preferably than intra- and/or inter-molecular viral genome recombination. In addition, this observation may have implication in the risk assessment of vector spillover into the liver.
S50
127. AAV-Mediated shRNA Integration into an Endogenous miRNA Locus as a New Strategy for Safe, Specific and Persistent Therapeutic RNAi
Stefan Mockenhaupt,1 Dirk Grimm.1 1 Infectious Diseases, Heidelberg University, Cluster of Excellence CellNetworks, Heidelberg, Baden-Wuerttemberg, Germany. The use of viral vector-delivered small hairpin RNAs (shRNAs) to induce RNAi-mediated gene silencing has started to revolutionize human gene therapy due to its efficiency, specificity and versatility. Yet, a critical prerequisite towards clinical translation remains the implementation of potent and safe means to prevent loss of the shRNA vector in proliferating infected cells. Ideally, this will be achieved via stable integration of the shRNA vector DNA into the human genome at a position tolerating insertions while providing sustained and regulated RNAi expression. Here, we accordingly propose sitespecific shRNA integration into an endogenous mi(cro)RNA locus as a novel strategy. For proof-of-concept, we engineered a plasmid to carry the authentic human miR-122 stem-loop structure along with 2.5 or 2 kb of flanking 5’ or 3’ genomic sequences, respectively. We next inserted a potent shRNA targeting human-alpha-1-antitrypsin (hAAT, a liver-borne enzyme) whose stem-loop mimics the miR122 structure into a position located about 100 bp upstream of miR-122 itself. Our hope and rationale were that following eventual integration into the genomic miR-122 locus, the endogenous miR122 promoter will permanently and efficiently co-transcribe the shRNA in a tissue-restricted fashion. Therefore, we first had to validate correct expression and functionality of both hairpin RNAs - ectopic shRNA and genuine miR-122 - encoded by our bicistronic sequence. We thus cloned the entire fragment under a strong viral RNA polymerase II promoter and measured knockdown of luciferase reporters tagged with binding sites for either small RNA. Indeed, we found that both target sequences were efficiently and specifically silenced upon expression of our chimeric plasmid, comparable to extents observed with individual sh or miRNA-encoding constructs. Encouraged by this, we have now transferred the entire fragment into an Adeno-associated viral (AAV) vector context and pseudotyped it with a new synthetic AAV capsid that we have recently engineered to mediate highly potent transduction of cultured cells. At present, we are using this vector in homologous recombination-based attempts to site-specifically integrate our chimeric sh/miRNA sequence into the endogenous miR-122 locus in a human liver cell line. Resulting clones will next be thoroughly characterized and scored positive based on a set of critical parameters, in particular unperturbed cell viability and correct concurrent sh/miRNA expression. Concomitantly, we will assess the potency and longevity of hAAT target gene silencing upon extended culture of single cellular clones. If successful, our approach opens up a wealth of novel avenues to safely integrate ectopic therapeutic shRNAs into human cells and to guarantee tissuespecific and persistent RNAi. We predict that by using tailored and potent viral capsids, and by targeting selected cell-specific miRNA loci, it will be feasible to expand our strategy to numerous clinically relevant organs and to apply the entire approach in vivo. Moreover, while we currently co-express a single shRNA, our construct can be readily modified to harbor multiple hairpins to permit combinatorial RNAi therapies.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy