- Email: [email protected]
967. Non-Viral Gene Transfer into Gunn Rats for Prevention of Drug Toxicity and Investigation of UGT Isoenzyme Function In Vivo
correction of fasting hypoglycemia and elevated long-chain acylcamitine species has been demonstrated. Over time (75 days) a loss of protein expression is seen with a gradual increase in long-chain serum acylcamitine species to near pre-treatment levels. Studies of hepatic DNA from early- and late-treated mice demonstrate a loss ofAAV genomes as one possible explanation for this. In an effort to develop a more durable expression vector with a more natural distribution oftissuc expression , we prepared several native VLCAD promoter constructs in a luciferase reporter system and compared in vitro promoter activity in 293 cells. Recombinant AAV2/8 vectors were then prepared using the two most promising promoter fragments (p 187. P1154). Following tail vein injection ofVLCADdeficient mice with these recombinant vectors, robust hVLCAD expression is seen in the liver and heart, and serum C 18acylcarnitine levels arc reduced to wild type levels by day II. Altogether, this work demonstrates the ability of the native hVLCAD promoter to produce sufficient hVLCAD protein to correct accumulated CI8 acylcamitine species in murine serum.
967. Non-Viral Gene Transfer into Gunn Rats for Prevention of Drug Toxicity and Investigation of UGT Isoenzyme Function In Vivo Istvan Danko, I Zhen Jia.'
'Department ofPediatrics, University ofWisconsin-Madison, Madison, IV/. Background: Variation in activ ity of UDP-glucuronosyl transferases (UGTs) in various tissues may be critical determinant of toxicological susceptibility. However overlapping substrate specificities make studying tissue-specific role of various UGTs in vivo very difficult. We have studied the feasibility ofnon-viral transfer of human and rat UGTs (hUGT, rUGT) into UGT deficient Gunn rats to conduct such studies. Methods: Animals: 4-6 week old Gunn and Wistar rats (GR, WR). Plasmid expression vectors for rUGTIAI , rUGTIA5, hUGTIAI were based on pCDNA3hUGTIAI containing CMV promoter (gill ofJ. Ritter). Control: pCI-LacZ (Promega, Madison, WI). Gene transfer: Seven days before Menadione injections groups ofGR received hepatic venous injection of 1/15 of body weight vector solution containing 0.6 mg of plasmid. Menadionc exposure and cvaluation of toxicitv: Serum alanine aminotransfcrase (ALl'; indicator ofhepatoeyte damage; UlL) was measured 24 hours after each ofthree consecutive daily intraperitoneal injections of various doses (5, 10, 15, 25, 50 mg/kg) of Menadione; serum bilirubin (SeBi J.lM/L) was measured before, and 7 and 10 days after gene transfer Gust before the first and one day after the last Menadione injection); Statistics: Data arc expressed as mean ± SO. Differences between the means from two independent samples werc tested with the t test. Results: Toxicitv of Menadione in WR vs. GR: Menadione dose of 10 mg/kg resulted in significantly higher ALl' levels in GR vs. WR (282±18 and 164.5±12 respectively, n""2, p<0.05) without mortality or significant morbidity by 24 hours after the second injection. At earlier time points and with < I0 mg/kg doses the difference was not significant. Mortality in GR was first observed at doses ~25 mglkg. Effect of hepatic expression of various UGTs on Menadione toxicity in GR: Serum ALl' 24 hours after the second Menadione injection of 10 rug/kg in GR expressing hUGTlA I was significantly lower than in GR not expressing hUGTIAI (158±24 vs. 282± 18; n=2 p<0.05). ALl' levels in GR expressing rUGT IA I, rUGTIA5 and lacZ 24 hours after the third injection of 15 rng/kg Menadione were 327.5±16, 433±28 and 416±1O respectively (significantly lower in rUGTIAI group vs. both other groups (n=4, p<0.05». Average SeBi before gene transfer, before first and one day after the third Menadione injection in the respective groups were: 124.8±23, 86.5±20, 93.5±1I; 121.9±21, 119±9.3, 114.5±12 and 120.9±20, 120.6±13, 117.5 ±IO. The decrease in serum bilirubin in GR expressing rUGTIAI vs. GR expressing rUGTIA5 or Molecular Therapy Volume15. Supplement I. ~ "'r 2007 Copyright © The Ameri can Soci ety o r Gene "1l1f:r:lpy
lacZ was statistically significant (n=4, p<0.05) Conclusions: UGT deficiency leads to increased Menadione toxicity. Non-viral expression of both human and rat UGTIAI in Gunn rat liver is protective against Menadione toxicity and leads to significant decrease in serum bilirubin. Selective expression ofUGTs in various tissues by non-viral gene transfer has great potential as a tool for investigating tissue-specific role ofvarious UGT isoenzymes and may provide an innovative gene therapy approach for prevention of the toxicity of various chemotherapy drugs and environmental toxins.
968. Patent Inventorship of Principal Investigators in Phase I Gene Transfer Clinical Trials: An Empirical Analysis Karen L. Durell,' Jonathan Kimmelman.' Josephine Nalbantoglu,' Richard Gold.'
'Faculty ofLaw, McGill University, Montreal, QC. Canada; 2Biomedical Ethics Unit, McGill University, Montreal, QC, Canada; 3 Department ofNeurology and Neurosurgery, McGill University, Montreal, QC, Canada. BACKGROUND: Policies of professional societies , institutions, and journals take widely varying positions on whether patent inventorship represents a potential interest conflict. Whereas institutions like the University of Pennsylvania and journals like JAMA have policies addressing patent inventorship, other universities (e.g. Washington University in St. Louis) and journals (e.g. Journal of Clinical Oncology) do not. Elsewhere, one of us OK) has argued that patent inventorship represents a financial interest that should be managed where human subjects are involved. PURPOSE: We undertook an empirical study to determine the frequency with which principal investigators in phase I gene transfer trials were named as inventors on patents relating to the study agent. METHODS: GEMCRIS entries and Appendix M filings were collected for 200 phase I gene transfer trials submitted to the OBA between 1999 and 2002. U.S. Patent and Trademark Office, and World Intellectual Property Organization Patent, Copyright and Trademark databases were searched using principal investigators in the inventor field. A certified patent agent (KD), in consultation with a gene transfer researcher (IN), determined whether patent elaims corresponded to any aspect ofthc investigational agent. After matches wcrc identified, we recorded the identity of the patent assignee, the identity of matching claims, and checked informed consent documents to determine whether patent interests were disclosed to research subjects. RESULTS: Among the various types of claims that matched protocols were: cells modified with specific genes, vectors, modes of agent administration, methods of expanding modified cells, treatment combinations, treatment steps, methods ofinducing physiological responses , and vector production processes. To date, patent records for 43 principal investigators (of 239 in our sample) have been sought. We found that 18 principal investigators (40%) were named as inventors on patents related to the study; this corresponded to eighteen protocols (45%) . Most patents (61%) were assigned to an institution hosting the clinical trial; the others were assigned to a principal invest igator. Of trials involving an investigator-inventor, 41% consent documents contained generic statements relating to commercialization. These generally took the form of statements about the usc ofdatabanked tissues. Disclosure of specific financial interests like patent invcntorship was observed for 17%. CONCLUSIONS: Our preliminary data indicate that inventorship is common in gene transfer trials. However, disclosure of patent inventorship in informed consent documents is sporadic. These findings, should they hold up after searching remaining protocols, have implications tor ethical review and informed consent practices (funding: Canadian Institutes of Health Research MOP-79288).
S369
967. Non-Viral Gene Transfer into Gunn Rats for Prevention of Drug Toxicity and Investigation of UGT Isoenzyme Function In Vivo Istvan Danko, I Zhen Jia.'
'Department ofPediatrics, University ofWisconsin-Madison, Madison, IV/. Background: Variation in activ ity of UDP-glucuronosyl transferases (UGTs) in various tissues may be critical determinant of toxicological susceptibility. However overlapping substrate specificities make studying tissue-specific role of various UGTs in vivo very difficult. We have studied the feasibility ofnon-viral transfer of human and rat UGTs (hUGT, rUGT) into UGT deficient Gunn rats to conduct such studies. Methods: Animals: 4-6 week old Gunn and Wistar rats (GR, WR). Plasmid expression vectors for rUGTIAI , rUGTIA5, hUGTIAI were based on pCDNA3hUGTIAI containing CMV promoter (gill ofJ. Ritter). Control: pCI-LacZ (Promega, Madison, WI). Gene transfer: Seven days before Menadione injections groups ofGR received hepatic venous injection of 1/15 of body weight vector solution containing 0.6 mg of plasmid. Menadionc exposure and cvaluation of toxicitv: Serum alanine aminotransfcrase (ALl'; indicator ofhepatoeyte damage; UlL) was measured 24 hours after each ofthree consecutive daily intraperitoneal injections of various doses (5, 10, 15, 25, 50 mg/kg) of Menadione; serum bilirubin (SeBi J.lM/L) was measured before, and 7 and 10 days after gene transfer Gust before the first and one day after the last Menadione injection); Statistics: Data arc expressed as mean ± SO. Differences between the means from two independent samples werc tested with the t test. Results: Toxicitv of Menadione in WR vs. GR: Menadione dose of 10 mg/kg resulted in significantly higher ALl' levels in GR vs. WR (282±18 and 164.5±12 respectively, n""2, p<0.05) without mortality or significant morbidity by 24 hours after the second injection. At earlier time points and with < I0 mg/kg doses the difference was not significant. Mortality in GR was first observed at doses ~25 mglkg. Effect of hepatic expression of various UGTs on Menadione toxicity in GR: Serum ALl' 24 hours after the second Menadione injection of 10 rug/kg in GR expressing hUGTlA I was significantly lower than in GR not expressing hUGTIAI (158±24 vs. 282± 18; n=2 p<0.05). ALl' levels in GR expressing rUGT IA I, rUGTIA5 and lacZ 24 hours after the third injection of 15 rng/kg Menadione were 327.5±16, 433±28 and 416±1O respectively (significantly lower in rUGTIAI group vs. both other groups (n=4, p<0.05». Average SeBi before gene transfer, before first and one day after the third Menadione injection in the respective groups were: 124.8±23, 86.5±20, 93.5±1I; 121.9±21, 119±9.3, 114.5±12 and 120.9±20, 120.6±13, 117.5 ±IO. The decrease in serum bilirubin in GR expressing rUGTIAI vs. GR expressing rUGTIA5 or Molecular Therapy Volume15. Supplement I. ~ "'r 2007 Copyright © The Ameri can Soci ety o r Gene "1l1f:r:lpy
lacZ was statistically significant (n=4, p<0.05) Conclusions: UGT deficiency leads to increased Menadione toxicity. Non-viral expression of both human and rat UGTIAI in Gunn rat liver is protective against Menadione toxicity and leads to significant decrease in serum bilirubin. Selective expression ofUGTs in various tissues by non-viral gene transfer has great potential as a tool for investigating tissue-specific role ofvarious UGT isoenzymes and may provide an innovative gene therapy approach for prevention of the toxicity of various chemotherapy drugs and environmental toxins.
968. Patent Inventorship of Principal Investigators in Phase I Gene Transfer Clinical Trials: An Empirical Analysis Karen L. Durell,' Jonathan Kimmelman.' Josephine Nalbantoglu,' Richard Gold.'
'Faculty ofLaw, McGill University, Montreal, QC. Canada; 2Biomedical Ethics Unit, McGill University, Montreal, QC, Canada; 3 Department ofNeurology and Neurosurgery, McGill University, Montreal, QC, Canada. BACKGROUND: Policies of professional societies , institutions, and journals take widely varying positions on whether patent inventorship represents a potential interest conflict. Whereas institutions like the University of Pennsylvania and journals like JAMA have policies addressing patent inventorship, other universities (e.g. Washington University in St. Louis) and journals (e.g. Journal of Clinical Oncology) do not. Elsewhere, one of us OK) has argued that patent inventorship represents a financial interest that should be managed where human subjects are involved. PURPOSE: We undertook an empirical study to determine the frequency with which principal investigators in phase I gene transfer trials were named as inventors on patents relating to the study agent. METHODS: GEMCRIS entries and Appendix M filings were collected for 200 phase I gene transfer trials submitted to the OBA between 1999 and 2002. U.S. Patent and Trademark Office, and World Intellectual Property Organization Patent, Copyright and Trademark databases were searched using principal investigators in the inventor field. A certified patent agent (KD), in consultation with a gene transfer researcher (IN), determined whether patent elaims corresponded to any aspect ofthc investigational agent. After matches wcrc identified, we recorded the identity of the patent assignee, the identity of matching claims, and checked informed consent documents to determine whether patent interests were disclosed to research subjects. RESULTS: Among the various types of claims that matched protocols were: cells modified with specific genes, vectors, modes of agent administration, methods of expanding modified cells, treatment combinations, treatment steps, methods ofinducing physiological responses , and vector production processes. To date, patent records for 43 principal investigators (of 239 in our sample) have been sought. We found that 18 principal investigators (40%) were named as inventors on patents related to the study; this corresponded to eighteen protocols (45%) . Most patents (61%) were assigned to an institution hosting the clinical trial; the others were assigned to a principal invest igator. Of trials involving an investigator-inventor, 41% consent documents contained generic statements relating to commercialization. These generally took the form of statements about the usc ofdatabanked tissues. Disclosure of specific financial interests like patent invcntorship was observed for 17%. CONCLUSIONS: Our preliminary data indicate that inventorship is common in gene transfer trials. However, disclosure of patent inventorship in informed consent documents is sporadic. These findings, should they hold up after searching remaining protocols, have implications tor ethical review and informed consent practices (funding: Canadian Institutes of Health Research MOP-79288).
S369