Christoph Volpers

Adenoviral vectors for gene transfer and therapy

Due to the very efficient nuclear entry mechanism of adenovirus and its low pathogenicity for humans, adenovirus‐based vectors have become gene delivery vehicles that are widely used for transduction of different cell types, especially for quiescent, differentiated cells, in basic research, in gene therapy applications, and in vaccine development. As an important basis for their use as gene medicine, adenoviral vectors can be produced in high titers, they can transduce cells in vivo with transgenes of more than 30 kb, and they do not integrate into the host cell genome. Recent advances in the development of adenoviral vectors have brought considerable progress on issues like target cell specificity and tropism modification, long‐term expression of the transgene, as well as immunogenicity and toxicity in vivo, and have suggested that the different generations of non‐replicative and replicative vectors available today will each suit best for certain applications. Copyright

Antibody-Mediated Targeting of an Adenovirus Vector Modified To Contain a Synthetic Immunoglobulin G-Binding Domain in the Capsid

ABSTRACT Adenovirus vectors have been targeted to different cell types by genetic modification of the capsid or by using recombinant or chemically engineered adaptor molecules. However, both genetic capsid modifications and bridging adaptors have to be specifically tailored for each particular targeting situation. Here, we present an efficient and versatile strategy allowing the direct use of monoclonal antibodies against cell surface antigens for targeting of adenovirus vectors. A synthetic 33-amino-acid immunoglobulin G (IgG)-binding domain (Z33) derived from staphylococcal protein A was inserted into the adenovirus fiber protein. The fiber retained the ability to assemble into trimers, bound IgG with high affinity (Kd = 2.4 nM), and was incorporated into vector particles. The transduction efficiency of the Z33-modified adenovirus vector in epidermal growth factor receptor (EGFR)-expressing cells was strongly and dose-dependently enhanced by combination with an EGFR-specific monoclonal antibody. The antibody-mediated increase in cellular transduction was abolished in the presence of competing protein A. In targeting experiments with differentiated primary human muscle cells, up to a 77-fold increase in reporter gene transfer was achieved by preincubation of the vector with monoclonal antibodies directed against neuronal cell adhesion molecule or integrin α7, respectively. The IgG-binding adenovirus vector holds promise for directed gene transfer to a wide variety of cell types by simply changing the target-specific antibody.

Targeting of High-Capacity Adenoviral Vectors

High-capacity adenoviral (HC-Ad) vectors contain only the noncoding termini of the viral genome, can deliver large DNA fragments of up to 36 kb into target cells, and feature reduced toxicity and prolonged transgene expression in vivo. To enhance the potential of HC-Ad vectors to transduce specific cell types, we constructed a versatile infectious new helper virus plasmid that can be used readily to introduce peptide ligands into the HI loop of the fiber knob domain of Ad5-based HC-Ad vectors. Helper viruses with a 6x-His epitope or Arg-Gly-Asp (RGD) peptide insertion retained the full infectivity of the wild-type helper virus. The RGD-modified helper virus was used for production of a capsid-modified HC-Ad vector expressing beta-galactosidase. The RGD HC-Ad vector transduced the ovarian carcinoma cell lines SK-OV-3 and OVCAR-3 with 4- to 20-fold higher efficiency, compared to unmodified vectors. Transduction of both primary vascular smooth muscle cells as well as primary human endothelial cells was increased up to 15-fold with the RGD-modified vector. Competition experiments with recombinant knob protein and different RGD peptides indicated that the RGD-mediated transduction was Coxsackie and Adenovirus receptor (CAR)-independent and involved integrin alpha(v)beta(5). The use of fiber-modified helper viruses in the last amplification step of HC-Ad vector production allows for convenient and efficient targeting of these vectors towards different cell types. Targeting strategies will increase the spectrum of applications for HC-Ad vectors and will further add to their safety.

Efficient and reproducible generation of high-expressing, stable human cell lines without need for antibiotic selection

BackgroundHuman cell lines are the most innovative choice of host cell for production of biopharmaceuticals since they allow for authentic posttranslational modification of therapeutic proteins. We present a new method for generating high and stable protein expressing cell lines based on human amniocytes without the requirement of antibiotic selection.ResultsPrimary amniocytes from routine amniocentesis samples can be efficiently transformed with adenoviral functions resulting in stable human cell lines. Cotransfection of the primary human amniocytes with a plasmid expressing adenoviral E1 functions plus a second plasmid containing a gene of interest resulted in permanent cell lines expressing up to 30 pg/cell/day of a fully glycosylated and sialylated protein. Expression of the gene of interest is very stable for more than 90 passages and, importantly, was achieved in the absence of any antibiotic selection.ConclusionWe describe an improved method for developing high protein expressing stable human cell lines. These cell lines are of non-tumor origin, they are immortalized by a function not oncogenic in human and they are from an ethically accepted and easily accessible cell source. Since the cell can be easily adapted to growth in serum-free and chemically defined medium they fulfill the requirements of biopharmaceutical production processes.

Strategies for muscle-specific targeting of adenoviral gene transfer vectors

Currently, adenoviral transfer of therapeutic genes such as dystrophin is hampered by low transduction efficiency of adult skeletal muscle. This is largely due to the lack of appropriate virus attachment receptors on the myofiber surface. Recent studies in transgenic mice revealed that upregulation of Coxsackie- and adenovirus receptor improves gene transfer efficiency by approximately ten-fold. Conversely, the vector load that needed to be administered to achieve sufficient gene transfer could be lowered significantly. Reduced viral vector loads may help to control virally mediated toxicity and immunogenicity. To date, there are no drugs or methods known to increase Coxsackie- and adenovirus receptor expression in skeletal muscle that would be easily applicable in humans. However, alternative strategies such as vector retargeting are currently being investigated that may allow for an increase in binding of adenoviral vectors to skeletal muscle. Recent experiments have shown that directed mutagenesis of the adenoviral fiber knob allows for a significant reduction in Coxsackie- and adenovirus receptor binding and for introduction of a new binding domain. Therefore, vector retargeting towards efficient and specific infection of skeletal muscle may be achieved by directed genetic alteration of adenoviral capsid proteins.

Human Cell Lines for Production of Biopharmaceuticals

Increasing demands for quality, identity and high expression rates have been the driving forces for the development of new expression systems over the last 10 years. In the future, human cell systems will range among leading technologies for expression and production of human therapeutic proteins. Primary human amniocytes obtained by routine amniocentesis have been immortalized by adenoviral E1/pIX functions. CEVEC’s Amniocyte Production (CAP) cells are of non-tumor origin, they are immortalized by a function not oncogenic in human, and they are from ethically accepted source of origin. Moreover, the development of CAP cells follows relevant guidelines and is completely documented. Starting from several hundred transformed cell lines, two CAP cell lines have been selected based on growth stability, performance in permanent and transient protein expression and capacity of growth in suspension. CAP cells grow as single cell suspension in chemically defined serum-free medium. CAP cells have been tested for permanent expression of reference proteins like human alpha-1 antitrypsin (hAAT), erythropoietin and human IgG1, very high protein expressing cell lines have been obtained. The hAAT expressed in CAP cell lines was fully glycosylated and sialylated. In addition, CAP cells show very high transfection efficiency, a major requirement for efficient transient expression of protein. Thus, we have developed a new expression system based on human amniocytes which offers significant advantages over existing production technologies, like a human glycosylation pattern or other post-translational modifications and high permanent and transient protein expression.

Viral Gene Transfer Into Endothelial Cells

Viral vectors are being employed to transfer genes into endothelial cells for experimental purposes in basic research as well as for gene therapeutic approaches for cardiovascular and oncological disorders. Therapeutic gene transfer strategies in the field of cardiovascular disorders are directed towards the stimulation of angiogenesis for ischemic heart disease, the inhibition of vascular smooth muscle cell proliferation, and functional protection of endothelial cells in order to prevent restenosis after angioplasty, the reduction of cholesterol blood levels for prevention of atherosclerosis, and the vasodilation or blockade of vasoconstriction in systemic and pulmonary hypertension (Khurana et al. 2001, Ehsan et al. 2000). The inhibition of endothelial cell proliferation and neoangiogenesis in tumors is a promising approach in oncological viral gene therapy developed in recent years.