Human Gene Therapy Vector 'GMP' Core Facility

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Human Gene Therapy Vector 'GMP' Core Facility

Summary

Drs. Nick Muzyczka [Director, UF Gene Therapy Center (UFGTC)], Terry Flotte [Co-Director, UFGTC], and Lung-Ji Chang [UFBI-funded UFGTC faculty member] and Ms. Kye Chesnut [Asst. Director, Research Services, UFGTC] attended the IFAB meeting in order to report on the UFGTC facilities and programs. Kye Chesnut was the main speaker. In keeping with the audience, special attention was given to the neuro-related gene therapy/delivery programs and to the Human Gene Therapy "GMP" Vector Core Laboratory (a.k.a. "Human Applications Lab") being constructed on the fifth floor of the new UFBI building.

Overview and rationale for UFBI funding the construction and equipping of the "GMP" lab.

Given the revolution in molecular medicine throughout the country, the number of different gene therapy applications currently active and envisioned by UF investigators (especially those involved in nervous system related research) and the encouraging results emerging from studies involving several different potentially therapeutic transgenes, there is a clear need for clinical grade vectors. However, to date, obtaining clinical grade vectors has been beyond the capabilities of most investigators, since the development, production scale, process testing and final product safety testing of these vectors is both technically difficult and prohibitively expensive. This, and the need for lot-to-lot consistency of preparations made under Good Manufacturing Practice (GMP) conditions, provided strong arguments in favor of the UFBI assisting in the establishment of a core facility for the production of clinical grade vectors. These and other considerations prompted Dr. Luttge to give the go ahead for the construction and equipping of a GMP lab (to be described below) for human gene therapy vector production on the fifth floor of the new building.

This new core lab will be managed and staffed by the UF Gene Therapy Center (UFGTC). It will operate in concert with the UFGTC's existing Preclinical Vector Core facility (located in rooms R4-140 and R-4-146 of the Academic Research Building) which, as restated below, serves to provide a platform for the development of procedures necessary for the production of large scale lots of clinical grade vector material. With these two core facilities, and the extraordinary technical expertise of the new and existing faculty and staff of the UFGTC (together with several planned recruitments), Dr. Luttge has expressed strong confidence and optimism that we should be able to handle the challenges of preclinical and clinical grade vector development for the UFBI, the rest of the UF Health Science Center, and any future collaborative interactions our faculty and students may have with DoD and DVA investigators.

UFGTC Mission Statement

The primary mission of the UFGTC is to merge molecular genetics research and health care delivery by developing new therapeutic strategies for the treatment of human diseases that involve gene transfer. Gene therapy is translational research and its success requires that high quality research programs be available in a variety of areas.

[Although the UFGTC has been in existence for only three years, Dr. Muzyczka and his colleagues have clearly already achieved phenomenal success in hiring new faculty (three to date), acquiring new extramural grant funding (e.g., two multi-million dollar NIH program project grants to date) and in building new core facilities (see below). One of the PO1 grants targets neuro gene delivery. The principal investigator of this grant is Dr. Nick Muzyczka; other key faculty include Drs. Paul Reier, Greg Schrimsher, Bill Hauswirth, Al Lewin and Sergei Zolotukhin.]

Functions of UF GTC Preclinical Vector Core Laboratory

Provide technical support, training, equipment, facilities and reagents for investigators to develop and test a range of first generation vectors intended for pre-clinical studies.

Design and test second generation vectors and to make technical improvements in the production of gene therapy vectors.

Provide a platform for the development of procedures necessary for the production of large scale lots of clinical grade vector material.

Develop and provide "in-house" quality control testing as necessary.

Functions of UFBI/UFGTC Human Applications Laboratory (GMP Facility)

Production of clinical grade vector material for Phase I/II gene therapy trials

Ex vivo manipulation/treatment of material for Phase I/II gene therapy trials

Description of the UFBI/UFGTC Human Applications Laboratory and Equipment

The UFBI/UFGTC Human Applications Laboratory (totalling approximately 1700 square feet) is divided into two completely separate facilities, the larger of which is designated for vector production, whereas the other is designated for ex vivo applications for gene therapy. Access to either side will be highly restricted with electronically-activated interlocking doors to assure proper, unidirectional human traffic flow through the facility. A high level of sterility and containment is maintained throughout the facility with HEPA-filtered supply and exhaust single pass air provided by an independent HVAC system, no running water or sinks, biological safety cabinets in which to handle the vectors and to aid in the production of Class 10,000 (Modules 1 and 2 on the production side) and Class 100,000 (Modules 3 and 4 on the ex vivo application side) clean rooms, and many other features. Much of the equipment will be the same for all four of the independent modules. Thus, in addition to the biological safety cabinets, each module will be equipped with two large capacity double stacked CO2 incubators for incubation of infected cells, an inverted microscope, low speed centrifuge (for harvest), and water bath (for warming media, etc.). In the support area surrounding the two modules on each side of the facility, there will be ultra low freezers for storage of materials and product, 6 foot double door refrigerators and standard freezers for storage of materials (media, serum, etc.). As the material will have to be qualified/certified prior to use, it is necessary to obtain and store large lots, and for these and other purposes the facility will have a cryogenic storage system to store cell lines as both master and working cell banks. The production side of the facility will also have the Cell Cube system to permit an easy linear scale-up production for anchorage-dependent cell lines. It will also have a microfluidizer for mechanical cell lysis and an HPLC for purification of the vectors. Each side of the facility has a computer at entry point to facilitate logging of activity, materials, environmental monitoring, etc.

Example Future Projects for the UFBI/UFGTC Human Applications Laboratory

In addition to a well developed human gene therapy program in cystic fibrosis being led by Dr. Terry Flotte, and the much newer programs on Pompe's (cardiomyopathy) disease and hemophilia being developed by Dr. Barry Byrne, there are several neuro-related gene therapy projects which may find there way to the UFBI/UFGTC Human Applications Lab in the not to distant future. One example is the autosomal dominant retinitis pigmentosa (ADRP) research program being led by Drs. Bill Hauswirth and Al Lewin. Twelve percent of American patients with this blinding disease carry a single amino acid substitution (P23H) in their rhodopsin gene resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Ribozymes have been developed that can discriminate and catalyze the destruction of these mutant RNAs in a transgenic rat model of ADRP when delivered to the eye via a recombinant adeno-associated vector (rAAV). The finding that ribozyme targeted destruction of P23H mutant RNA markedly slows the rate of retinal degeneration in transgenic rats, along with functional preservation of the retina, is the first demonstration of this therapeutic approach in an animal model of dominantly inherited human disease. In addition to this approach, Drs. Hauswirth and Lewin are also investigating rAAV as a means to deliver the growth factor, CNTF, to arrest degeneration in these same transgenic models. Patient populations are currently being identified for potential phase I trials.

A second example is the immunogene therapy approach for treating glioblastoma being led by Dr. Lung-Ji Chang. Glioblastoma is one of the most life-threatening cancers in the world. The most common procedure for treating this brain tumor is surgical removal followed by radiation therapy. Unfortunately, for most glioblastoma patients this therapy has tragically limited benefits. In fact, the average life expectancy for a high-grade glioblastoma patients is less than 6 months. One of the first gene therapy protocols approved for clinical trials was the retroviral vector mediated thymidine kinase (tk) gene transfer into brain tumor patients. The rationale for this treatment was that the tk gene transduced tumor cells were rendered sensitive to treatment with prodrugs such as gancyclovir. However, in spite of this positive outcome, Dr. Chang and his colleagues reasoned that the tk gene therapy for brain tumor would, at best, have only limited success because it was impossible to transduce all the tumor cells with the tk gene. In the past 4 years, in collaboration with Dr. Ken Petruk (Director of Neurosurgery at Univ. of Alberta), Dr. Chang has developed a novel gene therapy approach for treating glioblastoma patients in which a systemic anti-cancer immune response was induced using a combination of two immune modulatory genes: 1) the cytokine gene granulocyte-macrophage colony stimulating factor (GM-CSF), which enhances tumor antigen presentation; and 2) the T-cell co-stimulatory gene B7-2 (CD86), which activates cell-mediated immune responses. Using a novel human immune system reconstituted severe combined immunodeficiency (SCID) mouse/human glioblastoma model, Dr. Chang has successfully demonstrated that these two-gene modified tumor cells induced an anti-tumor immune response which prevented both de novo and previously established tumor cell growth. This study has resulted in a phase I gene therapy trial for treating brain and skin cancer patients. This trial has been approved by both the University of Alberta and the University of Florida cancer centers. The UF portion of this work will be done in collaboration with Dr. John Buatti.

Development of Lentiviral Vectors for Gene Transfer into Neural Tissues

One of the major obstacles in neural cell gene transfer is the lack of an optimal gene delivery vehicle to meet the following two criteria: efficient delivery and long term expression. In addition to the project described above, Dr. Lung-Ji Chang is also working on the development of lentiviral vectors suitable for neural cell gene transfer. He has already developed an HIV-based lentiviral vector with multiple safety features that eliminate the possibility of generating a replication competent virus. In preliminary studies he has shown that this vector transduces primary rat neurons and human neuronal stem cells at high efficiencies. Lentiviral vectors establish long term gene transduction due to the fact that chromosome integration is an essential step in the viral life cycle. Possible applications in neural tissue gene transfer with lentiviral vectors include gene therapy for Parkinson's disease (delivery of tyrosine hydroxylase gene), Alzheimer's disease (delivery of nerve growth factor), correction of other neuronal genetic defects and delivery of nerve growth factors for neural regeneration after injury. Dr. Chang is hoping for a close collaboration with other members of the UFBI in promoting these projects.