tbuckley/cabot-search huggingface dataset:
504. Restoration of Dystrophin Expression by Gene Editing with S. aureus Cas9 in Models of Duchenne Muscular Dystrophy
Authors: Jacqueline N. Robinson-Hamm, Christopher E. Nelson, Ruth M. Castellanos Rivera, Annemieke Aartsma-Rus, Aravind Asokan, Charles A. Gersbach
Journal: Molecular Therapy (2016)

Duchenne muscular dystrophy (DMD) is the most common fatal genetic disease characterized by progressive muscle wasting, loss of ambulation, and typically death in the third decade of life due to respiratory and cardiac complications. DMD results from deleterious mutations in the dystrophin gene that disrupts the translational reading frame and causes a complete lack of dystrophin protein. Becker muscular dystrophy (BMD) is similar to DMD in that it is the result of deletions in the dystrophin gene. However these deletions maintain the translational reading frame and result in the production of an internally truncated but partially functional dystrophin protein. The BMD phenotype is typically less severe than DMD, and thus converting DMD to a BMD-like phenotype by restoring the dystrophin reading frame is a widely explored therapeutic strategy. CRISPR/Cas9 can target precise loci to make specific DNA sequence changes in the genome. We have previously used S. pyogenes Cas9 (SpCas9) to restore dystrophin expression in immortalized myoblasts from DMD patients by deleting dystrophin exon 51 to repair the disrupted reading frame. A promising therapeutic application of this strategy involves in vivo viral delivery of the CRISPR/Cas9 system by AAV, however AAV cannot efficiently package the large SpCas9 gene along with full size promoters. Therefore we have made use of AAV delivery of the smaller S. aureus Cas9 (SaCas9) to delete exon 23 in the mouse dystrophin gene in vivo in the mdx mouse model of DMD and showed restored dystrophin expression and functional recovery. Here we are continuing this work by preparing a SaCas9 system targeted to the human dystrophin gene. gRNAs were designed to target the intronic regions flanking exon 51 and tested in vitro in HEK293T cells as well as immortalized DMD patient myoblasts that are correctable by removal of exon 51. The expected deletion was confirmed by PCR and sequencing of the genomic DNA and dystrophin cDNA. Western blot of lysates from differentiated cells confirmed restoration of dystrophin protein expression. We have also demonstrated exon 51 deletion in vivo following AAV delivery of this CRISPR/Cas9 system to the muscles of a mouse model containing the full length human dystrophin gene. Ongoing work involves testing this approach in a novel dystrophic mouse model carrying a mutated version of the human dystrophin gene that is correctable by exon 51 deletion. This work is important to the continued development of a translational strategy for gene editing as a potentially curative treatment for DMD.
Abstract 2247: SHP2 expression and function in different subtypes of glioblastoma
Authors: Laura Roccograndi, Yingtao Bi, Ramana Davuluri, Nadia Dahmane, Donald M. O'Rourke
Journal: Cancer Research (2015)

Abstract SHP2, a Src-homology 2 domain (SH2)-containing tyrosine phosphatase encoded by PTPN11, is a ubiquitously expressed cytoplasmic enzyme that regulates events downstream of several growth factor and cytokine receptors involved in survival, pro-migratory and differentiation signaling. In the normal developing nervous system, SHP2 is involved in neural stem cell self renewal. Dysregulation of SHP2 function has been implicated in tumorigenesis and characterized as an activated downstream regulator of oncogenes in gastric carcinoma, anaplastic large-cell lymphoma, breast cancer and glioblastoma. In the context of glioblastoma, we have shown that SHP2, and specifically its phosphatase activity, is directly involved in EGFRvIII-induced oncogenesis. Our present research aims at understanding the function of SHP2 within different glioma cell types as well as the four characterized subtypes of glioblastoma (GBM). Genomic analysis of SHP2 expression using the TCGA GBM cohort shows differential expression among the recently characterized subtypes of GBMs, with higher SHP2 mRNA expression in the classical subtype. Additionally, SHP2 is preferentially expressed in glioma stem cells versus differentiated cells isolated from the same patients. Furthermore, our current analysis shows that reduction of SHP2 expression in glioma stem cells affects the expression of genes linked to stemness. SHP2 PTPase function may therefore be a link between normal stem cell proliferation and gliomagenesis induced by EGFR/RTK activated signaling. Citation Format: Laura Roccograndi, Yingtao Bi, Ramana Davuluri, Nadia Dahmane, Donald M. O'Rourke. SHP2 expression and function in different subtypes of glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2247. doi:10.1158/1538-7445.AM2015-2247
Donor CTLA-4 Gene Polymorphism Associations with Acute GvHD After Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Irena Frydecka, Lidia Karabon, Edyta Pawlak-Adamska, Anna Tomkiewicz, Tomasz Wrobel, Miroslaw Markiewicz, Slawomira Kyrcz-Krzemien
Journal: Blood (2011)
Hepatocellular Carcinoma Is the Most Common Indication for Liver Transplantation and Placement on the Waitlist in the United States
Authors: Ju Dong Yang, Joseph J. Larson, Kymberly D. Watt, Alina M. Allen, Russell H. Wiesner, Gregory J. Gores, Lewis R. Roberts, Julie A. Heimbach, Michael D. Leise
Journal: Clinical Gastroenterology and Hepatology (2016)
679. Efficient Reconstitution of a 7kb Therapeutic Mini-Dystrophin Gene in Duchenne Muscular Dystrophy Dog Muscle
Authors: Kasun Kodippili, Xiufang Pan, Hsiao Yang, Chady Hakim, Yadong Zhang, Yongping Yue, Dongsheng Duan
Journal: Molecular Therapy (2015)

Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in the dystrophin gene. Restoring dystrophin expression by adeno-associated virus mediated gene therapy holds great promise as a mutation-independent therapy for all DMD patients. The full-length dystrophin cDNA (≈11 kb) is far beyond the 5 kb packaging capacity of a single AAV vector. Consequently, truncated micro- and mini-dystrophin genes have been used. The micro- and mini-dystrophin genes encode ≈30% and ≈50% of the full-length dystrophin coding sequence, respectively. Mini-dystrophin is expected to provide better protection than micro-dystrophin. We have previously shown that dual AAV vectors can efficiently express a 6 to 8-kb mini-dystrophin in mdx mice. Dual AAV-mediated mini-dystrophin gene therapy significantly ameliorated histopathology and improved muscle function in mdx mice, a mouse model of DMD. To translate this promising therapy to large mammals, here we evaluated the reconstitution of a 7-kb minigene in the canine model of DMD by local injection. We engineered a pair of dual-AAV vectors to express a 7-kb canine codon-optimized ΔH2-R15 mini-dystrophin gene. For easy detection, a flag-tag and a GFP gene were fused to the N-terminal and C-terminal ends, respectively. To determine whether dual AAV vectors can lead to efficient mini-dystrophin expression, we co-delivered both vectors to the extensor carpi ulnaris muscle in the forelimb of DMD dogs at a dose of 2×013 vg particles/vector/muscle. Two months after gene transfer we evaluated transduction efficiency and observed successful expression of mini-dystrophin from the dual-AAV vectors. All flag-tag positive myofibers were also positive for minidystrophin, GFP and dystrophin-associated glycoprotein complex proteins. Importantly, mini-dystrophin gene therapy also reduced muscle force loss under the stress of repeated cycles of eccentric contraction. These results establish the proof-of-concept for mini-dystrophin gene therapy in dystrophic muscles of large mammals.
528. Generation of a Cocal Envelope Packaging Cell Line for Robust Lentiviral Gene Transfer Into Hematopoietic Stem Cells and T Cells
Authors: Olivier M. Humbert, Amie B. Adams, Donald W. Gish, Martin E. Wohlfahrt, Jennifer E. Adair, Grant D. Trobridge, Hans-Peter Kiem
Journal: Molecular Therapy (2015)

Lentiviral vectors (LVs) are routinely used for stable gene transfer and have demonstrated great promise in clinical hematopoietic stem cell and immune cell gene therapy. LVs are commonly pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G), which confers broad tropism to the vector and allows for vector concentration by centrifugation. However, the use of VSV-G has several limitations, such as susceptibility to inactivation by human serum complement making it unsuitable for in vivo delivery, and toxicity when constitutively expressed, which has impeded efforts to generate stable producer cell lines. With the goal to generate a self-inactivating LV packaging cell line, we stably expressed either the VSV-G or cocal envelope in HEK293T cells along with other required helper genes, 3rd generation gagpol and rev. Genes were sequentially introduced in cells by co-transfection with plasmids containing an antibiotic resistance gene. After selection, best producer clones were isolated by limiting dilution and were treated with the histone deacetylase inhibitor sodium butyrate to enhance vector titer. The resulting cocal packaging cell line produces 20 to 50-fold more infectious particles as compared to VSV-G expressing cells, reaching titers averaging 108 infectious units/mL upon concentration. We also extend on our previous studies (Trobridge, 2010) by showing more robust gene transfer for cocal-pseudotyped LVs as compared to VSV-G pseudotyped LVs in human (81% cocal vs. 45% VSV-G, MOI 5) and non-human primate (Macaca nemestrina) CD34+ HSPCs (45% cocal vs. 18% VSV-G, MOI 2×5) while preserving similar differentiation potential as determined by CFC assays. Cocal vectors also transduced human CD3+ blood cells more efficiently than VSV-G vectors (65% cocal vs. 42% VSV-G vs., MOI 3) and showed comparable transduction efficiency in non-human primate CD3+ cells. Using a competitive repopulation approach in the non-human primate model, we find increased gene marking in repopulating cells transduced with a cocal pseudotyped LVs compared to repopulating cells transduced with the same lentivirus backbone pseudotyped with VSV-G. We observed a dramatic difference in the granulocyte fraction where marking with the cocal vector reaches ~90% at 70 days post transplantation. Retroviral integration site analysis is currently underway to characterize the integration profile of cocal vs. VSV-G modified clones. Overall, our study suggests that the cocal envelope outperforms the VSV-G envelope in a stable lentivirus packaging cell line, which may prove useful in current gene therapy efforts to generate large-scale clinical grade vectors.
Prognostic Significance of CXCR4 Overexpression in Pediatric Acute Myeloid Leukemia with Low-Risk: A Report from the Japanese Pediatric Leukemia/Lymphoma Study Group
Authors: Hidemasa Matsuo, Naomi Nakamura, Daisuke Tomizawa, Akiko M. Saito, Nobutaka Kiyokawa, Keizo Horibe, Yoko Nishinaka-Arai, Mayu Tokumasu, Hiroshi Itoh, Yasuhiko Kamikubo, Hideki Nakayama, Akitoshi Kinoshita, Takashi Taga, Akio Tawa, Tomohiko Taki, Shiro Tanaka, Souichi Adachi
Journal: Blood (2015)
417. Induction of Potent Cytotoxic and Antitumor Activity By a Highly Optimized hTERT DNA Vaccine
Authors: Jian Yan, Jaemi S. Chu, Nyamekye Obeng-Adjei, Matthrew P. Morrow, Kimberly Kraynyak, Anna M. Slager, Maria Yang, Amir S. Khan, Kate E. Broderick, Niranjan Y. Sardesai, David B. Weiner
Journal: Molecular Therapy (2015)

Immunotherapy of cancer through induction of anti-tumor cellular immunity has recently re-emerged as an important therapy for the treatment of nonresponsive cancers. Human telomerase reverse transcriptase (hTERT) is highly expressed in more than 85% of human tumors, with little or no expression in normal somatic cells. Expression of hTERT correlates well with telomerase activity and is associated with tumor cell growth and contributes crucially to the long-term survival of tumor cells. In addition, targeting hTERT may have the potential to eliminate cancer stem cells as recent studies have suggested that cancer stem or stem-like cells express hTERT. Immunological analysis supports that hTERT is a widely applicable target recognized by T cells and can be potentially studied as a broad cancer immune therapeutic, or as a unique line of defense against tumor recurrence. There remains an urgent need to develop more potent hTERT vaccines. Here, we developed a synthetic highly optimized hTERT DNA vaccine (phTERT) to express full-length hTERT. When delivered by electroporation, phTERT elicited robust hTERT-specific CD8 response (the total response in phTERT-immunized mice was 1817±211 SFU/106splenocytes). Epitope mapping results showed that 9 out of 26 matrix pools exhibited more than 50 spots, indicating phTERT could elicit a broad range of T-cell immune responses. The average percentage of CD107a, IFN-γ and TNF-α secreting cells from the total CD8+ T cell population were approximately 0.8%, 1.2% and 1.8%, respectively. Moreover, vaccination with phTERT slows the tumor growth in HPV16-associated tumor-bearing mice. Six out of ten phTERT-immunized mice still survived thirty-nine days post tumor implantation while all mice in naïve group were dead or euthanized. In vivo cytotoxicity assay confirmed that phTERT-induced CD8 cells exhibited specific CTL activity and were capable of eliminating hTERT-pulsed target cells. In addition, the ability of phTERT to overcome tolerance was evaluated in a NHP model, whose TERT protein is 96% homologous to that of hTERT. Immunized monkeys exhibited robust (average of 1834 SFU/106PBMCs), diverse (multiple immunodominant epitopes) IFN-γ responses and antigen-specific perforin release (average of 332 SFU/106 PBMCs), suggesting phTERT breaks tolerance and induces potent cytotoxic responses in this human relevant model. Lastly, we demonstrated that co-immunization with a rhesus macaque IL-12 plasmid as an adjuvant could further increase the vaccine-induced responses for about two fold in NHP. Taken together, these findings support that this synthetic EP-delivered DNA phTERT may have a role as a broad therapeutic cancer vaccine candidate.
Abstract 2336: AD1 promotes astroglial differentiation and depletes glioblastoma stem cells
Authors: Raffaella Spina, Dillon M. Voss, Andrew Sloan, Eli E. Bar
Journal: Cancer Research (2015)

Abstract Glioblastoma are among the least curable cancers in man, at least in part because of stem-like cellular subpopulations (herein referred to as glioma stem cells, GSC) refractory to current therapies. The cancer stem cell hypothesis suggests that tumor cells are organized in a pyramidal unidirectional differentiation cascade with GSC at the top and functionally defined by the ability to self-renew and initiate tumors identical to the original tumors from which they are derived. GSCs are maintained by both cell intrinsic and micro-environmental factors and conditions. In this study we focused on identifying small molecules which target tumor heterogeneity by promoting differentiation of GSC into less aggressive, more differentiated subpopulations. To this end, we have developed reporter models for astroglial and neuronal differentiation in HSR-GBM1, HSR040622 and HSR040821 tumor-derived, GSC-enriched, neurospheres lines, using lineage-specific reporter constructs. We employed these reporter lines to screen a library of over 700 bioactive small molecules for potential inducers of GSC differentiation. We identified several molecules which significantly induce astroglial differentiation in multiple GSC-enriched lines. In this report, we focus on one of these agents, (AD1 = (Astroglial Differentiation-1), which induces astroglial differentiation by up to 30-fold as compared with DMSO control. Interestingly, the effects of AD-1 appear to be primarily cytostatic rather than cytotoxic. As differentiation should result in reduced self-renewal capacity, we next determined the effect of AD1 on clonogenicity in vitro using the extreme limited dilution assay (ELDA). We found that 48h treatment with AD1 significantly reduced clonogenic capacity of HSR-GBM1, HSR-040622 and HSR-040821 reporter lines by over 90%. To test this in vivo we examined the effect of AD1 on tumor initiation in an intracranial transplantation model. We found that ex vivo treatment with AD1 resulted in a significant increase in the survival of animals as 6 (of 6) animals transplanted with DMSO-treated cells exhibited massively infiltrating tumors while 2 (of 6) of the animals implanted with AD1 treated cells developed tumors, and those began to appear several weeks later. Importantly, the effects of AD1 are likely to be irreversible as cells extracted from tumors maintained reporter expression even after six passages in vitro. Taken together, our results support the therapeutic value of a drug-induced differentiation and define how differentiation affects the GSC compartment in glioma. Citation Format: Raffaella Spina, Dillon M. Voss, Andrew Sloan, Eli E. Bar. AD1 promotes astroglial differentiation and depletes glioblastoma stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2336. doi:10.1158/1538-7445.AM2015-2336
Abstract 3637: Delivery of an epigenetic agent using a microparticle drug delivery system
Authors: Duc P. Do, Keane O. Soluade
Journal: Cancer Research (2015)

Abstract Conventional cancer treatment protocols are either toxic or of minimal therapeutic value, necessitating investigation of new modalities for efficacious treatment and management of cancers. Among the various novel compounds, histone deacetylase inhibitors (HDACIs) target cancer cells over-expressing deacetylases (HDACs). The inhibition of HDACs changes gene expression without altering the DNA sequence. In the present study, an albumin-based microparticulate drug delivery system containing vorinostat was formulated and evaluated for breast cancer therapy. Drug-loaded microparticles were prepared by a microencapsulation method through spray drying. Prepared microparticles containing the active drug were characterized for their physiochemical properties, including size, surface morphology, Zeta potential and surface properties. Dissolution studies were carried out to examine the release profile of the drug from the delivery system. For in vitro characterization and efficacy studies, breast carcinoma HTB-126 wells were used. Histone deacetylase (HDAC) activity was determined from cancer cells, and the efficacy of the free vorinostat and microparticle formulations was compared. Global histone changes in cells were analyzed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Western blot was also utilized to detect acetylation marks in cells. Uptake studies were carried out in cancer cells to examine the internalization capability of microparticle drug formulations. Data indicated that microparticles were approximately 2-μm in size with Zeta potential measurements of about -30 mV. Release studies in cells showed that the drug released from the microparticulate delivery system at an extended rate over a period of over 24 hours. In vitro data in cancer cell line indicated that there was at least 10% more inhibition of cell viability when vorinostat was used with the microparticles. Similar results were observed from clonogenic studies. Additionally, in vitro data suggested that cell death induced by the drug-loaded microparticles was caused by apoptosis. Drug-loaded microparticle formulations were able to be internalized into cancer cells. Microparticles were shown to be a promising targeting strategy for cancer epigenetic therapy by providing an extended-release of the drug in the body. Citation Format: Duc P. Do, Keane O. Soluade. Delivery of an epigenetic agent using a microparticle drug delivery system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3637. doi:10.1158/1538-7445.AM2015-3637
PTU-084 Should UK gastroenterologists be trained in transcutaneous liver biopsy?: Abstract PTU-084
Authors: B J Colleypriest, K Fagan, P F Marden
Journal: Gut (2010)

<h3>Introduction</h3> The UK Joint Committee of Higher Medical Training Gastroenterology curriculum (Feb 2005) suggests that specialist registrars should be “skilled in liver biopsy”. The Joint Royal College of Physicians Training Board Speciality curriculum for gastroenterology (May 2007) states that “liver biopsy is mandatory for those trainees doing liver subspecialty, however other trainees may have and wish to take the opportunity to learn this technique either while doing their hepatology training or at other points in their training rotation”. Increasingly liver biopsy is performed under ultrasound guidance, which has subsequently reduced trainees9 exposure to the technique. In 2005 we surveyed trainee gastroenterologists and demonstrated a low level of practical experience and theoretical knowledge of transcutaneous liver biopsy.<sup>1</sup> The aim of this study was to establish whether these deficiencies had improved. <h3>Methods</h3> All gastroenterology SpRs and ST trainees in the Wessex deanery received a questionnaire aimed at assessing practice training in liver biopsy and knowledge of the current BSG guidelines. The results of the survey were compared to previously published data from 2005. <h3>Results</h3> 66% of the 31 gastroenterology trainees surveyed responded. Similar to 2005, all of the trainees worked in hospitals where liver biopsies were performed exclusively under ultrasound guidance by radiologists. 43% (compared with 54% in 2005) had performed liver biopsy with supervision and only 19% (29% in 2005) were independently competent. Only 19% of trainees felt that either gastroenterologists or herpetologists should be performing transcutaneous liver biopsy. The average number of liver biopsies performed per trainee had dropped from 14.1 in 2005 to 9.7 in 2009. Only 13% of trainees had performed liver biopsy without supervision; a significant drop from 39% in 2005. None of the trainees experienced in liver biopsy had a written record of this. Knowledge of the suggested safe platelet count, INR and complication rates were: <h3>Conclusion</h3> This survey of gastroenterology trainees demonstrates a low level of practical and theoretical knowledge with respect to transcutaneous liver biopsy. These findings concur with data from 2005 and in fact reveal a decline in the number of liver biopsies performed by trainees and independent competency. If training in liver biopsy is to remain mandatory for hepatology trainees and recommended for gastroenterologists, training in ultrasound is necessary and training should be aligned with that undertaken by radiologists.
Phase II Studies of Different Schedules and Doses of the Farnesyl Transferase Inhibitor Tipifarnib (R115777, Zarnestra, NSC-702818) for Patients of Age 70 or Older with Previously Untreated Acute Myeloid Leukemia (AML): A North American Intergroup Study (S0432).
Authors: Harry P. Erba, Kenneth J. Kopecky, Mark H. Kirschbaum, Martin S. Tallman, Richard A. Larson, Cheryl L. Willman, Marilyn L. Slovak, Holly M. Gundacker, Frederick R. Appelbaum
Journal: Blood (2007)