340 A Multi Use GMP Facility for Cell Processing in an Academic Center Designed for the Implementation of New Regulatory Requirements Molecular Therapy �������� ��� ���� ���������������� �������� ����[.]
Trang 1Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts
CANCER TARGETED GENE THERAPY I would provide new insights into the mechanism of tumor angiogenesis
in general and may have the potential for the development of novel
anti-angiogenic gene therapy approaches for prostate cancer in
particular
a Potential Therapy for Cancer
Rana S Al-Assah, Rachel L Cowen, Edwin C Chinje, Kaye J
Williams, Brian A Telfer, Ian J Stratford
1 Experimental Oncology Group -Pharmacy Department,
Manchester University, Manchester, United Kingdom.
Tumor growth is a complex phenomenon regulated by a variety
of molecules including Nitric Oxide (NO) NO is produced during
the conversion of L-arginine to citrulline, which is catalyzed, by the
Nitric Oxide Synthase (NOS) family of enzymes in particular
inducible NOS (iNOS) The role of NO in tumor growth remains
controversial: low levels promote tumor growth and angiogenesis
while high levels can be cytotoxic through the induction of apoptosis
we have shown that like P450Reductase, iNOS can metabolise
pro-drugs like tirapazamine (TPZ) Thus, because of the great potential
for iNOS as a therapeutic in a gene therapy approach, we have
harnessed iNOS expression using the ecdysone inducible system
This will allow us to tightly control the levels of iNOS expression
and exclude its unwanted pro-tumor effects
The inducible system was first tested by generating stable
HT-1080 clones (human fibrosarcoma) expressing inducible lac Z Low
basal levels and high fold induction in response to Ponasterone A
achieved in vitro Also, this clone was grown in nude mice which
were later injected with Pon A (1mg/mouse) After staining with
X-gal, xenografts of the clone turned blue throughout Following this
evaluation, stable clones of HT-1080 encoding for inducible iNOS
expression were generated This clone has been fully evaluated (max
pmole/min/mg without Pon A) and in vivo(6.57 pmole/min/mg with
1 mg Pon A /mouse compared to undetectable levels in uninduced
tumors) Using this xenograft model we are studying the effects of
inducible iNOS expression on tumor vascularity and the impact of
this on tumor growth and bioreductive drug distribution Moreover,
adenoviruses encoding for iNOS expression (lac Z) in the ecdysone
cassette have been generated allowing us to extend our work to a
variety of cell-lines We aim to use these viruses to induce high
levels of iNOS expression in vitro and in vivo allowing us to pulse
high levels of NO and induce apoptosis In addition these tumor
cells will be sensitised to killing by TPZ and radiation
Human Airway Epithelial Cells Via Arginine-Rich
Polypeptides
James Lausier,1 Kevin Foley,1 Emanuela Bruscia,1 Wolfgang
Dostmann,1 Dieter C Gruenert.1
1 Departments of Medicine and Pharmacology, University of
Vermont, Burlington, VT.
One obstacle to transfecting DNA into eukaryotic cells is the
delivery of DNA to the nucleus This feature of DNA transfection
has important implications for in vitro gene expression and in vivo
gene therapy Numerous DNA delivery systems, both viral and
non-viral, have been developed to overcome this obstacle with varying
degrees of success A number of non-viral systems have been based
on cationic polypeptides This study evaluates the potential of
arginine rich-polypeptides (DT-5 and DT-6) to facilitate transfer
and release of DNA in the nucleus or human airway epithelial cells
These polypeptides are based of sequences derived from the HIV
Tat protein and have been shown to facilitate both trans-cytoplasmic and trans-nuclear transport of nucleic acids To test the potential of this DNA delivery system to facilitate transport of small DNA fragments (SDF) into the nucleus of these cultured epithelial cells, SDF were mixed at different charge ratios of peptide to DNA Gel retardation analysis showed that the complexes were stable After delivery into the cells, confocal analysis of differentially labeled DNA (red fluorescence) and peptide (green fluorescence) demonstrated that the SDF were transferred to the nucleus within 4-8 hrs and then release within the nucleus as free DNA Further studies are underway to evaluate whether the SDF can mediate modifications in genomic DNA both in vitro and in vivo
Processing in an Academic Center - Designed for the Implementation of New Regulatory
Requirements
Gerhard Bauer,1 Jon E Walker,1 Jan A Nolta,1 Steven Devine,1 John F DiPersio.1
1 Oncology, Washington University School of Medicine, St Louis,
MO, United States.
Introduction: Over the recent years clinical applications of cellular therapy, particularly gene therapy, have come under much scrutiny Many academic centers, until recently, have only used dedicated lab spaces or even only dedicated biosafety cabinets to perform clinical grade tissue manipulations This kind of practice is prone to contamination, particularly when an open system (flask system) for tissue culturing is applied New and stricter regulatory requirements have recently been established for researchers who want to translate laboratory research into Phase I and II clinical trials These new regulations already mandated or to be mandated in the near future by the regulatory agencies will require many academic centers to rethink their approach towards clinical trials of gene - or cellular therapy, and even tissue processing for transplantation Objective: To be in compliance with current Good Laboratory Practice (GLP), Good Tissue Practice (GTP), and Good Manufacturing Practice (GMP), Washington University School of Medicine in St Louis undertook the difficult task of designing and constructing a new GMP laboratory for cellular therapy Six Class 10,000 manufacturing labs are the core of this 2,615 square foot facility Class 100,000 intermediate exit and entry rooms separate the manufacturing rooms from other areas, and serve as the only access for the individual Class 10,000 labs There is no personnel or air exchange directly from one manufacturing lab to another This makes the facility completely versatile in terms of production of separate cellular products without cross-contamination from personnel or air Personnel flow is unidirectional only, from a gowning room into an intermediate entry room, from intermediate entry room into one manufacturing lab at one time only To exit, personnel enters an intermediate exit room and proceeds into the de-gowning room The highest air pressure is maintained in the manufacturing rooms, while the gowning areas have negative air pressure towards the hallway This prevents contaminants from the outside air to enter the manufacturing rooms, and protects the outside environment from contamination with material that will be manufactured in the GMP facility Since one of the major requirements of a GMP facility
is to operate it under GMP conditions, a strict Quality Control and Quality Assurance program has been put in place Environmental cleaning and monitoring is addressed in a range of Standard Operating Procedures (SOPs) written specifically for this GMP lab Summary: A GMP facility must provide an environment for the safe processing and manipulation of tissues for use in humans under the new and stricter regulations Its versatility in an academic center
Trang 2Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
S134
CANCER TARGETED GENE THERAPY I
should allow for new protocols to be developed and readily applied
in the ever changing and more demanding field of cellular and gene
therapy
DNA for Pharmaceutical Applications: Current
Plasmid Quality Standards, Contaminations,
Process Difficulties and Solutions
Markus Mueller,1 John Vu,2 Wayne Tvrdik,2 Astrid Breul,1
Joachim Schorr.1
1 QIAGEN GmbH, Hilden, Germany; 2 QIAGEN,Inc., Valencia,
CA, United States.
A prerequisite for the use of plasmid DNA in clinical trials for
gene therapy and genetic vaccination is a manufacturing process
that is suitable to generate the pharmaceutical product in a
“state-of-the-art” quality Also, such a manufacturing process needs to
meet the requirements concerning batch scale, robustness,
reproducability and regulatory compliance
As the efficacy of a (plasmid) drug determines the commercial
success or failure of the complete project, the feasability of the
manufacturing process has a similar impact
This presentation will illustrate the plasmid quality standards as
they are currently accepted for use in clinical trials by regulatory
authorities around the world Critical contaminations and their causes
will be discussed and the challenge of the removal thereof These
considerations will be done viewing on pilot to large scale
manufacturing processes Knowing the hurdles, preventive actions
can be implemented in the manufacturing to improve the quality of
the plasmid product used in clinical trials and therewith enhancing
the probability to succesfully bring a product from the research and
clinical phases into the commercial market
Intraperitoneal Administration in
Measles-Susceptible Mice
Rae Myers,1 Marie Frenzke,1 Suzanne Greiner,1 Mary Harvey,1
Diane Soeffker,1 Evanthia Galanis,2 Kah-Whye Peng,3 Stephen
Russell.1,3
1 Toxicology Core, Mayo Foundation, Rochester, MN; 2 Medical
Oncology, Mayo Foundation, Rochester, MN; 3 Molecular
Medicine Program, Mayo Foundation, Rochester, MN.
MV-CEA is an attenuated oncolytic measles virus engineered to
express the soluble extracellular domain of human carcinoembryonic
antigen Viral gene expression can be followed noninvasively in
MV-CEA treated tumor bearing animals by monitoring the plasma
concentrations of CEA Based on the promising antitumor efficacy
of MV-CEA in a human ovarian xenograft model, we have developed
a phase I/II clinical protocol in which we propose to test the agent
in patients with advanced, treatment refractory ovarian cancer
Increasing doses of MV-CEA, harvested by lysis of virus-infected
Vero cells, will be administered by intraperitoneal infusion to
successive cohorts of three patients until dose-limiting toxicity is
encountered In support of the proposed clinical study we have
evaluated the toxicity of the agent in CD46 receptor-transgenic,
interferon receptor knockout mice that were previously shown to
be susceptible to measles virus infection Groups of 6 to 8 week old
mice were challenged intraperitoneally with a single dose of
MV-CEA (10e5, 10e6, or 10e7pfu) or with six doses of 10e7 pfu
administered over a two-week period Control mice were either
uninjected or injected with Vero cell lysate containing no virus
Animals were individually microchipped and monitored regularly
out to 90 days for changes in general wellbeing, coat condition,
activity level, body weight, plasma chemistry profile, complete
blood count and blood coagulation Plasma CEA values were determined to confirm that active MV-CEA virus had been administered and to elucidate the kinetic profile of viral gene expression Major organs were harvested and examined histologically for acute (day 4), subacute (day 21) and chronic (day 90) toxicity
We found that intraperitoneal administration of Vero cell lysate is almost completely innocuous in the CD46 transgenic mice, and the toxicities seen (all minor) were due primarily to MV-CEA administration The toxicities that were documented included transient abnormalities in biochemical and hematological parameters
of alkaline phosphatase, BUN, total protein, and WBC count, as well as a slowed growth rate seen only in high single and multiple dose groups There was no evidence of peritonitis due to intraperitoneal administration of vehicle or virus at any dose level All organs examined, except for the spleen, appeared normal at every time point in every group In the early time points for the high single and multiple dose groups, we saw splenomegaly of 2-3 times normal size with microscopic findings of follicular hyperplasia with a pronounced increase of immunoblasts at the borders of the B cell follicles and focally infiltrating into the red pulp Late time points and lower dose groups did not show any abnormalities We
do not anticipate significant toxicities associated with MV-CEA in our proposed clinical study
Measles, Mumps, and Vaccinia Vectors Against Ovarian Cancer Xenografts
Rae Myers,1 Marie Frenzke,1 Suzanne Greiner,1 Mary Harvey,1 Diane Soeffker,1 Katalin Abraham,2 Alan Shaw,2 Shmuel Rozenblatt,3 Kah-Whye Peng,4 Stephen Russell.1,4
1 Toxicology Core, Mayo Foundation, Rochester, MN; 2 Merck Research Laboratories, West Point, PA; 3 Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv, Israel;
4 Molecular Medicine, Mayo Foundation, Rochester, MN.
We directly compare several gene therapy vectors against an in vivo intraperitoneal model of ovarian SKOV3ip.1 tumor in order to look at the relative survival time and thus the efficacy of the various viruses, as well as the dose-responsiveness of MV-CEA-treated tumor bearing mice
MV-CEA is an attenuated oncolytic measles virus engineered to express the soluble extracellular domain of human carcinoembryonic antigen Viral gene expression can be followed noninvasively in MV-CEA treated tumor bearing animals by monitoring the plasma concentrations of CEA MV-Moraten is the Moraten vaccine strain
of measles virus produced by Merck MVA-H/F is a recombinant modified vaccinia virus Ankara (MVA), encoding the measles virus (MV) hemagglutinin (H) and fusion (F) glycoproteins The mumps virus is a Jeryl-Lynn vaccine strain paramyxovirus Prior to the start of the study, all mice were microchipped and ear-notched to ensure accurate identification Female 5 week old nude mice were challenged intraperitoneally with 3.5E6 SKOV3ip.1 cells day 0, with treatment administered beginning day 7 by the same route Groups of 10 female mice were treated with (1) 10E7 pfu x 1 MV-CEA, (2) 10E7 pfu x 6 MV-MV-CEA, (3) 1.75 x 10E5 pfu x 6 MV-MV-CEA, (4) 1.75 x 10E5 pfu x 6 MV-Moraten, (5) 10E7 pfu x 1 MVA-H/F, (6) 2 x 10E8 pfu x (7) 2 x 10E8 x 1 MVA, (8) 10E7 pfu x 1 Mumps,
or (9) neat x 6 Vero cleared cell lysate control, and were monitored for survival MV-CEA treated mice had blood drawn periodically
to obtain a CEA profile At necropsy, total intraperitoneal tumor burden and location was recorded All measles virus and mumps virus treated groups (MV-CEA, MV-Moraten, and Mumps) showed prolonged survival as compared to the vehicle control Survival was not prolonged in vaccinia virus treated groups (MVA and MVA-H/ F) and significant acute treatment-related toxicity was observed in these animals Survival of measles and mumps treated animals was