- Email: [email protected]
265. Nanoparticles with Triplex-Forming Oligonucleotides for Site-Specific Editing of the Human Cystic Fibrosis Transmembrane Receptor Gene
OLIGONUCLEOTIDE & RNAI THERAPEUTICS I into VSMCs but not ECs. To identify candidate aptamer clones, deep-sequence data from all rounds of selection were analyzed using novel bioinformatics methods. These methods include: (1) metrics of selection enrichment, (2) pairwise comparisons of edit and tree distance and (3) correlation analyses of single aptamer changes between rounds (e.g. fold change). Importantly, we demonstrate the efficacy and specificity of these aptamer clones in arterial segments ex vivo. Finally, single aptamers were conjugated to siRNAs that silence genes (cell cycle regulators, redox genes) implicated in promoting VSMC proliferation and migration following vascular injury. We show that silencing of several redox genes (e.g. NADPH oxidase subunits) results in reduced proliferation and migration of VSMCs in culture. The aptamer-siRNA conjugates are currently being evaluated for efficacy and safety in murine models of vascular injury. In conclusion, we have developed novel methodologies for identifying cell-specific, cell-internalizing RNA aptamers. Future refinement of these methodologies will enable the identification of aptamers to a multitude of different cell types for the broad development of targeted cell therapies.
264. Phase I Study of pbi-shRNA™ STMN1 Bilamellar Invaginated Vesicle (BIV) Lipoplex (LP) Via Intratumoral Injection in Advanced Cancer
John Nemunaitis,1,2,3,4 Donald D. Rao,4 Nancy S. Templeton,4 Neil Senzer,1,2,3,4 Zhaohui Wang,4 Minal Barve,3 Padmasini Kumar,4 Gladice Wallraven,4 Joseph Kuhn,5 Peter Beitsch,2 Phillip B. Maples.4 1 Mary Crowley Cancer Research Centers, Dallas; 2Medical City HCA, Dallas; 3Texas Oncology, P.A., Dallas; 4Gradalis, Inc., Dallas; 5WLS Surgical Associates, P.A., Dallas. STMN1 is critically involved in control of microtubule dynamics. Knockdown of STMN1 as a single agent and in combination with microtubule stabilizing chemotherapy (e.g. taxanes) result in increased G2M phase cell population and apoptosis. Using a miR30scaffold we developed a novel bi-shRNA strategy which demonstrates more effective silencing of target gene expression than same target shRNA or siRNA by concurrently inducing translational repression and mRNA sequestration in the p-body as well as post-transcriptional mRNA cleavage/degradation (Rao et al., Ca Gene Ther 2010). Testing in in vitro and in vivo STMN1 expressive tumor models demonstrated improved response and survival correlating with STMN1 knockdown. Moreover, toxicology and biodistribution testing in mice and biorelevant rats support Phase I clinical evaluation. IND application has recently been approved by FDA/RAC for Phase I testing of pbishRNA™ STMN1 LP to be administered as a single IT injection in patients with accessible refractory cancer. Patients will accrue in 4-patient escalation cohorts using a modified Fibronacci escalation schema (100%→50%→33%→33%) at a starting intratumoral dose of 0.010 mg/kg of DNA through a dose of 0.053 mg/kg DNA intratumoral / single dose. Should a single, but not more than two (2), ≥ Grade 3 Dose Limiting Toxicity (DLT) occur, an additional two (2) patients will be accrued at that dose (total of six). Serum for pharmacokinetics (PK) will be collected at multiple time points after study agent administration. One week prior to the injection, a biopsy of the accessible lesion will be obtained. Subsequently, using a randomized approach, a biopsy of the injected lesion will be obtained at 24 hours post-injection in 2 patients and at 48 hours post-injection in the remaining 2 patients per cohort. Tissue samples will be tested for RT-qPCR, 5’ RLM-RACE assay to detect STMN1 mRNA cleavage and qPCR for quantitation of plasmid presence (Davis, Zuckerman et al. 2010). All lesions will be excised or, if not appropriate, incisionally biopsied on Day 7 post-injection for IHC, H&E, plasmid, and RT-qPCR assays. Steps in engineering of the IND application process, as well as preliminary patient response results will be presented. S104
265. Nanoparticles with Triplex-Forming Oligonucleotides for Site-Specific Editing of the Human Cystic Fibrosis Transmembrane Receptor Gene Nicole A. McNeer,1 Kavitha Anandalingam,1 Rachel Fields,1 Christina Caputo,1 Peter M. Glazer,1 Marie Egan,1 William M. Saltzman.1 1 Yale University School of Medicine, New Haven, CT.
Cystic fibrosis (CF) is an autosomal recessive disorder caused by the production of a defective cystic fibrosis transmembrane receptor (CFTR) protein, resulting in respiratory and digestive dysfunction. CF affects 70,000 patients worldwide, with the median age of survival of affected individuals in the late 30s. The most common mutation in CF is a three base-pair deletion (Δ508) resulting in the loss of a phenylalanine residue. One way to combat this monogenic disorder is through site-specific gene editing, which would correct the faulty gene at its endogenous site. Triplex-forming peptide nucleic acids (PNAs) can be used to mediate the homologous recombination of short 50-60 base-pair “donor DNA” fragments, resulting in specific gene modification. We have designed triplex-forming PNA molecules and donor DNA targeting the CFTR gene, and use polymer nanoparticles to deliver these oligonucleotides for gene editing in human CF bronchial epithelial cells. We tested several tail-clamp PNA molecules for binding to the targeted site in the CFTR gene. We found that one of these molecules, when delivered with donor DNA in poly(lactide-co-glycolide) (PLGA) nanoparticles, results in correction of the Δ508 mutation in human CF bronchial epithelial cells. Correction occurred in ∼1% of cells following a single treatment, and modification was confirmed by direct sequencing and a chloride flux assay. There was no detectable modification in an off-target site with homology to the PNA molecule. We have recently shown that PLGA nanoparticles can be used to mediate direct in vivo editing of human genes in various tissues, including in the lung, after systemic injection. This provides a strong rationale for the use of these triplexPNA containing nanoparticles for in vivo editing of the CFTR gene. Our approach is unique in that it combines triplex and polymer nanoparticle technologies for safe, specific editing of CFTR.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
264. Phase I Study of pbi-shRNA™ STMN1 Bilamellar Invaginated Vesicle (BIV) Lipoplex (LP) Via Intratumoral Injection in Advanced Cancer
John Nemunaitis,1,2,3,4 Donald D. Rao,4 Nancy S. Templeton,4 Neil Senzer,1,2,3,4 Zhaohui Wang,4 Minal Barve,3 Padmasini Kumar,4 Gladice Wallraven,4 Joseph Kuhn,5 Peter Beitsch,2 Phillip B. Maples.4 1 Mary Crowley Cancer Research Centers, Dallas; 2Medical City HCA, Dallas; 3Texas Oncology, P.A., Dallas; 4Gradalis, Inc., Dallas; 5WLS Surgical Associates, P.A., Dallas. STMN1 is critically involved in control of microtubule dynamics. Knockdown of STMN1 as a single agent and in combination with microtubule stabilizing chemotherapy (e.g. taxanes) result in increased G2M phase cell population and apoptosis. Using a miR30scaffold we developed a novel bi-shRNA strategy which demonstrates more effective silencing of target gene expression than same target shRNA or siRNA by concurrently inducing translational repression and mRNA sequestration in the p-body as well as post-transcriptional mRNA cleavage/degradation (Rao et al., Ca Gene Ther 2010). Testing in in vitro and in vivo STMN1 expressive tumor models demonstrated improved response and survival correlating with STMN1 knockdown. Moreover, toxicology and biodistribution testing in mice and biorelevant rats support Phase I clinical evaluation. IND application has recently been approved by FDA/RAC for Phase I testing of pbishRNA™ STMN1 LP to be administered as a single IT injection in patients with accessible refractory cancer. Patients will accrue in 4-patient escalation cohorts using a modified Fibronacci escalation schema (100%→50%→33%→33%) at a starting intratumoral dose of 0.010 mg/kg of DNA through a dose of 0.053 mg/kg DNA intratumoral / single dose. Should a single, but not more than two (2), ≥ Grade 3 Dose Limiting Toxicity (DLT) occur, an additional two (2) patients will be accrued at that dose (total of six). Serum for pharmacokinetics (PK) will be collected at multiple time points after study agent administration. One week prior to the injection, a biopsy of the accessible lesion will be obtained. Subsequently, using a randomized approach, a biopsy of the injected lesion will be obtained at 24 hours post-injection in 2 patients and at 48 hours post-injection in the remaining 2 patients per cohort. Tissue samples will be tested for RT-qPCR, 5’ RLM-RACE assay to detect STMN1 mRNA cleavage and qPCR for quantitation of plasmid presence (Davis, Zuckerman et al. 2010). All lesions will be excised or, if not appropriate, incisionally biopsied on Day 7 post-injection for IHC, H&E, plasmid, and RT-qPCR assays. Steps in engineering of the IND application process, as well as preliminary patient response results will be presented. S104
265. Nanoparticles with Triplex-Forming Oligonucleotides for Site-Specific Editing of the Human Cystic Fibrosis Transmembrane Receptor Gene Nicole A. McNeer,1 Kavitha Anandalingam,1 Rachel Fields,1 Christina Caputo,1 Peter M. Glazer,1 Marie Egan,1 William M. Saltzman.1 1 Yale University School of Medicine, New Haven, CT.
Cystic fibrosis (CF) is an autosomal recessive disorder caused by the production of a defective cystic fibrosis transmembrane receptor (CFTR) protein, resulting in respiratory and digestive dysfunction. CF affects 70,000 patients worldwide, with the median age of survival of affected individuals in the late 30s. The most common mutation in CF is a three base-pair deletion (Δ508) resulting in the loss of a phenylalanine residue. One way to combat this monogenic disorder is through site-specific gene editing, which would correct the faulty gene at its endogenous site. Triplex-forming peptide nucleic acids (PNAs) can be used to mediate the homologous recombination of short 50-60 base-pair “donor DNA” fragments, resulting in specific gene modification. We have designed triplex-forming PNA molecules and donor DNA targeting the CFTR gene, and use polymer nanoparticles to deliver these oligonucleotides for gene editing in human CF bronchial epithelial cells. We tested several tail-clamp PNA molecules for binding to the targeted site in the CFTR gene. We found that one of these molecules, when delivered with donor DNA in poly(lactide-co-glycolide) (PLGA) nanoparticles, results in correction of the Δ508 mutation in human CF bronchial epithelial cells. Correction occurred in ∼1% of cells following a single treatment, and modification was confirmed by direct sequencing and a chloride flux assay. There was no detectable modification in an off-target site with homology to the PNA molecule. We have recently shown that PLGA nanoparticles can be used to mediate direct in vivo editing of human genes in various tissues, including in the lung, after systemic injection. This provides a strong rationale for the use of these triplexPNA containing nanoparticles for in vivo editing of the CFTR gene. Our approach is unique in that it combines triplex and polymer nanoparticle technologies for safe, specific editing of CFTR.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy