Gene therapy, tissue engineering of skin and blood vessels, controlled protein and gene delivery

Stelios T. Andreadis, Associate Professor

Research in my group focuses in three areas: tissue engineering, gene therapy and functional genomics. Tissue engineering holds promise for an entirely new approach to the repair and reconstruction of tissues/organs damaged by disease, injury or genetic abnormalities.   Another promising therapeutic modality, gene therapy, is broadly defined as the transfer of genes to cells/tissues for a therapeutic effect. This new technology has the potential to revolutionize tissue engineering by providing the means to impart new functions or enhance existing ones in a tissue/organ substitute. In addition, high throughput technologies such as cDNA and protein arrays may play an important role in tissue engineering to obtain the molecular “fingerprint” of engineered tissues and understand tissue development and tissue integration after implantation in vivo.

Tissue Engineering

Genetically modified skin equivalents for wound healing

In my group we develop model systems to study the process of wound healing and re-epithelialization. We developed an in vitro model of wound reepithelialization based on skin equivalents of human keratinocytes to evaluate biomaterials as substrate for keratinocyte growth and migration after wounding and as vehicles for the delivery of wound healing factors. We also developed an in vivo model of wound healing, which is based on tissue-engineered skin transplanted onto nude mice. A few weeks post-transplantation the implant is wounded and the wounds are treated with biomaterials carrying genes or proteins. Since skin equivalents are based on human cells, this model allows us to study healing of human tissue in an animal. We also use recombinant retrovirus to genetically modify epidermal keratinocytes in order to (i) promote wound healing for patients with genetic skin defects, burns or injuries; and (ii) mark cells in these in vitro grafts, in order to study the process of growth, differentiation and migration under normal conditions or after injury.

Biomaterials for controlled delivery of gene and proteins for wound healing

We seek to design biomaterials to promote keratinocyte migration and as vehicles for the delivery of genes and proteins to promote wound healing. Initially, we evaluate these biomaterial formulations in vitro using the three-dimensional model of wound healing that we developed. The optimum formulations are then tested in nude mice transplanted with human skin equivalents, in order to obtain data that are relevant to the more complex process of wound healing and at the same time minimize the number of animal experiments.

Vascular Tissue Engineering

More recently we engineered small diameter vessels, which exhibit considerable mechanical strength and reactivity to multiple constrictors (e.g. KCl, U46619, norepinephrine) and dilators (e.g. SNAP) after only two weeks in culture. We are currently studying the response of these tissues to pulsatile forces and testing their behavior in vivo using sheep as an animal model. This model system can be used as a biological model to address interesting questions with regards to vessel development and disease progression and has the potential to be used for treatment of vascular disease. As such, our work may have significant impact in cardiovascular tissue engineering.

Gene Therapy

Retrovirus gene transfer using extracellular matrix molecules

Gene therapy is a new therapeutic modality that is defined as the transfer of genetic material to cells/tissues to achieve a therapeutic effect. Several technologies for gene transfer exist, but to date recombinant retroviruses are used in the majority of gene therapy clinical trials. Our work aims at developing systems to achieve high efficiency of retroviral transduction and study the physicochemical properties of recombinant retroviruses. We have recently shown that optimization of key physicochemical factors improved immobilization of retrovirus to fibronectin and increased gene transfer by an order of magnitude. We have also discovered that retrovirus binds to fibronectin through heparan sulfate proteoglycans, which it may acquire from the virus producer cells. These findings may have important implications for gene therapy and for understanding virus cell interactions.

Retrovirus purification

Based on our understanding of the interactions of retrovirus with extracellular matrix we developing a novel method to purify recombinant retrovirus and designed a reactor to improve the efficiency and scale up this process. Such developments are necessary in order to realize the transition of genetic engineering from the bench to the clinic.

Gene transfer to epidermal stem cells

Although retroviral transduction results in permanent genetic modification of target cells, differentiation and eventually loss of the transduced cells from the epidermis results in temporary transgene expression. Our work aims at increasing gene transfer to the cells with the highest growth potential, namely the keratinocyte stem cells, in order to achieve high efficiency, targeted and long-term expression of the transgene. We discovered that retroviral gene transfer depends on expression and function of integrins on the surface of epidermal keratinocytes. In addition, long-term experiments suggest that retroviral transduction on extracellular matrix results in preferential gene transfer to epidermal stem cells. We are currently working to understand the role of integrins at the molecular level and prove transduction of stem cells through long term in vivo transplantation of genetically modified skin equivalents. Our findings may have important implication for gene transfer to epidermal stem cells and they may also find wider applicability to stem cells of other tissues.

Functional Genomics

Functional Genomics in Tissue Engineering

The three dimensional tissues that we construct are also used as model systems to study tissue development. The complex nature of tissue differentiation with the numerous molecular players involved calls for methods that allow the rapid and simultaneous quantitation of a large number of genes that participate in the process. Toward this end, we employ the technology of cDNA arrays to monitor the levels of gene expression during epidermopoiesis in vitro and in vivo. Specifically, we have used cDNA arrays to study the response of engineered skin to barrier disruption and the protective effects of keratinocyte growth factor. This study revealed very interesting molecular information with regards to the response of engineered tissue to injury and helped us develop novel hypotheses to understand how complex molecular interactions lead to a certain tissue phenotype. We propose that functional genomics can be used to obtain the molecular fingerprint of engineered tissues before application in the clinic or as cell biosensors.

Applications of nanoparticles in functional genomics and gene therapy

Nanotechnology is an emerging field that has the potential to transform medicine, biotechnology, manufacturing, energy, and information processing, as recognized by the various nanotechnology initiatives being promoted by federal funding agencies. The concepts and tools of nanotechnology can be applied to a large number of bioengineering applications including tissue engineering and gene therapy. To this end we have initiated an interdisciplinary project at the interface of nanotechnology and bioengineering (with M.T. Swihart). This project aims at using nanoparticles with unique optical properties to develop novel high throughput assays and facilitate delivery and detection of genes to target cells for the purpose of gene therapy. In parallel, we develop mathematical models to study the biophysics of surface DNA hybridization in order to understand how kinetic and transport limitations may affect the accuracy and sensitivity of on DNA microarrays (with Prof. Johannes Nitsche).