Benitec Biopharma Ltd (BNTC)

[georgejjl]

Gradalis and Strike Bio inc.

[535] In Vivo Study of a Bi-Functional Short-Hairpin RNA (bi-shRNA) Targeting Multiple Single-Nucleotide KRAS Mutations in Pancreatic Cancer

Donald D. Rao, Chris M. Jay, Zhaohui Wang, Padmasini Kumar, Neil Senzer, John Nemunaitis. Gradalis, Inc., Dallas, TX; Mary Crowley Cancer Research Centers, Dallas, TX; Strike Bio, Inc., Dallas, TX

KRAS is a member of guanine nucleotide-binding protein family and acts as a key molecular switch regulating multiple intracellular signaling pathways including epidermal growth factor receptor (EGFR). KRAS mutations essentially result in constitutively active KRAS and unregulated downstream signaling. The majority of pancreatic ductal adenocarcinoma (PDAC) patients carry a single-nucleotide KRAS mutation, >90% of which are single amino acid substitutions of Glycine at codon 12 in exon 2. The five-year survival rate of PDAC is less than 5%. KRAS mutation-specific targeting remains elusive and recent small molecule therapeutics demonstrates weak affinity or rapid degradation. We have recently developed a novel bi-functional shRNA (bi-shRNA) RNA interference (RNAi) technology that achieves in vitro selective knockdown of mutant KRAS expression (Figure 1).

We systematically determined the relative normal/mutant allele discriminating knockdown activity of a series of constructs by sequential evaluation of guide strand position effect. The single nucleotide mutation at positions 2-5 of the guide strand to be the most effective. Similar constructs were made and tested for G12D, G12V, G12R (comprising 91% of PDAC KRAS mutations) and G12C mutations (2.9%). G12D and G12C selective bi-shRNA expression vectors did not inhibit HEK-293 (KRASwt) growth in contrast to cell growth reduction of PANC-1 (KRASwt/G12D) and MiaPaCa2 (KRASwt/G12C). By adopting the miR-17-92 polycistronic miRNA backbone, multiplex knockdown targeting G12D, G12C and G12R were determined to be effective for multiple expressive KRAS mutant alleles with a single vector construct. A second multiplex construct targeting G12C, G12D and G12V achieved similar results. Mice are a biorelevant species for the triple bi-shRNA KRAS lipoplex. Preliminary safety testing revealed no toxic effect in vivo following a single intravenous dose of liposome encased bi-shRNA KRAS triplexes. In vivo efficacy studies with PANC-1 or MIAPaCa-2 pancreatic cancer models will be reported. Application of this therapeutic formulation with combination to EGFR targeting agents will also be examined.
Keywords: Non-Viral Gene Delivery; Oligonucleotide Based Therapies; RNAi and shRNA

Session: Simultaneous Oral Abstract Sessions-Mini Session: RNAi (3:15 PM-4:15 PM)
Date/Time: Friday, May 23, 2014 - 4:00 PM
Room: Washington 4

http://www.abstracts2view.com/asgct/view.php?nu=ASGCT14L1_535

G-CON’s autonomous POD™ provides process space for Strike Bio’s bifunctional RNA technology platform for personalized medicine

There is no other solution on the market or that can be built that allows us to have the proper environment with the ability to seamlessly scale up and expand cGMP manufacturing operations in the timeframe needed and for the cost of the POD.
College Station, TX (PRWEB) September 19, 2014

G-CON Manufacturing, Inc., the manufacturer of autonomous cleanroom PODs™, is pleased to announce that Strike Bio, Inc. has selected G-CON to provide an ISO 7 cleanroom POD to further advance its specific protein knockdown bifunctional short hairpin RNA (shRNA) technology. Strike’s proprietary bi-functional shRNA expression platform has been developed to target driver genes and interacting dominant pathway and co-pathway nodes to inhibit tumor growth. This technology has been shown to be effective in several cancer therapies currently in the clinical trial process.
Building on those successes, Strike Bio is purchasing an 18’W x 42’L x 11.5’H (5.49m x 12.8m x 3.51m) standard POD for further production and development of the bifunctional technology. The POD provides approximately 468 sq.ft. (43.5 sq.m.) of cleanroom space, with a 108 sq.ft. (10.0 sq.m.) area for gowning and equipment pass through. G-CON will also provide the shipping, delivery, installation, and commissioning of the POD.
“Strike Bio is a rapidly growing company with ambitious plans for expansion as we continue to advance our platform from preclinical through clinical trials. Our selection of the POD to be able to further our technology was an easy one. There is no other solution on the market or that can be built that allows us to have the proper environment with the ability to seamlessly scale up and expand cGMP manufacturing operations in the timeframe needed and for the cost of the POD,” said Dr. Donald Rao, Director of Interference Technologies.
“We are excited to provide a POD to an innovative company such as Strike,” said Maik Jornitz, President of G-Con Manufacturing, Inc. “Personalized medicine is the future and Strike’s technology is at the forefront of this movement. Given Strike’s expectation that it will scale up rather quickly, the POD was really the only solution that would work for Strike.”
The sale of this POD is the first for G-Con in the personalized medicine field, a field that is expected to grow exponentially in the coming years. In anticipation of such growth, G-Con has launched its scalable 9’W x 50’L miniPOD CT Series which includes a built in corridor system and unidirectional flow layout. Such PODs can be utilized individually or in parallel as part of a POD system.

http://www.prweb.com/releases/2014/09/prweb12180140.htm

[115] Pilot: Coordination of Patient Relevant Genome Identified Targets and Triple Targeting bi-shRNAi Nanoplex for Clinical Assessment in Advanced Renal Cell Cancer (RCC)

John Nemunaitis, Neil Senzer, Joseph Kuhn, David Shanahan, Alyssa Roth, Zhaohui Wang, Padmasini Kumar, Xiuquan Luo, F. C. Brunicardi, Chris M. Jay, Donald D. Rao. Mary Crowley Cancer Research Centers, Dallas, TX; Texas Oncology, P.A., Dallas, TX; Medical City Dallas Hospital, Dallas, TX; Gradalis, Inc., Dallas, TX; Strike Bio, Inc., Dallas, TX; WLS Surgery Associates, P.A., Dallas, TX; Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA

RCC is uniquely characterized by inactivation of the VHL gene and consequent VHL complex. We characterized multiple primary site samples of malignant and non-malignant tissue from a patient with newly diagnosed metastatic RCC utilizing Next Generation sequencing to perform whole exome sequencing and RNA sequencing (SeqWright, Houston, TX). Quality deep sequencing data were used for multiple bioinformatics analysis; detection for variants (GATK-1.4), and annotated (SnpEff-3.0), copy number variation (NextGENe-v2.3.3 from SOFTGENETICS Inc.), structure variation (BreadDancer-1.0), gene differential expression (Cufflinks-2.1.1). A S111R and S29R mutations and deletion of VHL gene, CDKN2A/B deletion, FH deletion, LOH at the BAP1, PBRM1 and SETD2 cluster and MYC (c-Myc) amplification were identified. Several gene expression changes were observed at the mRNA level, among those up regulated genes that are related to cancer survival/progression are SLC2A1 (GLUT1), FLT1 (VEGFR1), TGFB1 (TGFß1), TGFA (TGFa), MYC (c-Myc), HIF1A (HIF1a), CA9 (CAIX), SLC16A4 (MCT4), and HK2. The data correlated well with data generated by FoundationOne service using DNA recovered from paraffin embedded tissues and further validated at protein level by western immunoblots.
bi-shRNA interference encoding clinical grade DNA vectors were designed, constructed and verified within two months to each of these signals. Cell lines (RCC4, 786-0, CAKI-1 and G-401) that contain similar signal defects are being utilized to define optimal vector activity. Utilizing miR 17-92 cluster sequence backbone previously described involving other targets (KRAS, PDX-1, STMN1), a triplet, "GMP" (clinical) grade bi-shRNA multiplex based on network analysis and demonstrating the most active antitumor response will be constructed and incorporated in a previously described liposome nanoplex [Phadke et al DNA and Cell Biology 2011; Liu et al PLoS ONE 2012] for IND establishment consistent with other bi-shRNA IND's (BB-IND 14938; BB-IND 14205) necessary for experimental clinical management.
Keywords: Cancer Gene Therapy; Gene Correction/Modification; RNAi and shRNA

Session: Poster Session: Gene Regulation and Targets (5:30 PM-7:30 PM)
Date/Time: Wednesday, May 21, 2014 - 5:30 PM
Room: Hall A and B South

http://www.abstracts2view.com/asgct/view.php?nu=ASGCT14L1_115

Good luck and GOD bless,

George