An Approach to Achieve Long-Term Expression in Skin Gene Therapy

Therrien, Jean-Philippe; Pfützner, Wolfgang; Vogel, Jonathan

doi:10.1177/0192623307312705

Cited by 4 publications

(3 citation statements)

References 58 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For example, small number of keratinocytes can be isolated, expanded in monolayer culture, and developed into tissue sheets for autologous epidermal replacement of regions with extensive burns or ulcers (33). Keratinocyte-mediated gene therapy is another intensively studied area of research, whereby therapeutic genes can be introduced into autologous keratinocytes and regrafted onto the host (34).…”

Section: Figurementioning

confidence: 99%

Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor

Chapman

Meyers³

et al. 2010

J. Clin. Invest.

282

314

View full text Add to dashboard Cite

Primary human keratinocytes are useful for studying the pathogenesis of many different diseases of the cutaneous and mucosal epithelia. In addition, they can form organotypic tissue equivalents in culture that can be used as epidermal autografts for wound repair as well as for the delivery of gene therapy. However, primary keratinocytes have a finite lifespan in culture that limits their proliferative capacity and clinical use. Here, we report that treatment of primary keratinocytes (originating from 3 different anatomical sites) with Y-27632, a Rho kinase inhibitor, greatly increased their proliferative capacity and resulted in efficient immortalization without detectable cell crisis. More importantly, the immortalized cells displayed characteristics typical of primary keratinocytes; they had a normal karyotype and an intact DNA damage response and were able to differentiate into a stratified epithelium. This is the first example to our knowledge of a defined chemical compound mediating efficient cell immortalization, and this finding could have wide-ranging and profound investigational and medical applications. IntroductionSomatic cells have a limited lifespan, gradually slow in growth, and stop dividing, a process known as cellular senescence. This process is thought to limit the vulnerability of aging cells to disease. Human keratinocytes are invaluable for the study of skin biology and the pathogenesis of skin-related diseases, but their short lifespan in culture is a limitation. Different conditions have been developed to optimize the culture of keratinocytes; for example, the presence of fibroblast feeder cells increases the proliferative capacity of primary keratinocytes from approximately 20 to 40-60 population doublings (1, 2). Spontaneously immortalized keratinocyte lines, for example HaCaT (3) and NIKS (4), have been used for skin-related research. However, these cell lines have genetic abnormalities, such as mutations in p53 (5) or isochromosomes (4).Continuous replication of primary human cells is blocked by 2 separate events: mortality stage 1 (M1; replicative senescence) and mortality stage 2 (M2; crisis). At M1, signaling by shortened telomeres results in activation of the p53 and pRB pathways. M2 represents a critical period of genomic instability, with extremely eroded telomeres, resulting in chromosomal fusions and translocations. In retinal pigment epithelial cells and foreskin fibroblasts (which have decreased expression of components of the p16INK4A/pRB pathway), telomerase expression is sufficient to bypass both M1 and M2 and stabilize and elongate chromosome ends (6). However, telomerase expression is not sufficient for immortalization of keratinocytes, and p16INK4A function must also be disrupted (7,8).Keratinocytes expressing the E6 and E7 proteins encoded by high-risk human papillomavirus (HPV) types bypass both M1 and M2 blocks and become immortal (reviewed in ref. 9). E7 inactivates and degrades the pRB retinoblastoma tumor suppressor protein to induce G 1 /S phase progression of th...

show abstract

Section: Figurementioning

confidence: 99%

Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor

Chapman

Meyers³

et al. 2010

J. Clin. Invest.

282

314

View full text Add to dashboard Cite

show abstract

“…Skin is an excellent target for gene therapy [10-15] due to its easy accessibility for delivery and monitoring. GET of skin is a simple and direct method for gene therapy and can be accomplished in a minimally invasive way.…”

Section: Introductionmentioning

confidence: 99%

Thermal Assisted In Vivo Gene Electrotransfer

Donate¹,

Bulysheva²,

Edelblute³

et al. 2016

CGT

View full text Add to dashboard Cite

Gene electrotransfer is an effective approach for delivering plasmid DNA to a variety of tissues. Delivery of molecules with electric pulses requires control of the electrical parameters to achieve effective delivery. Since discomfort or tissue damage may occur with high applied voltage, the reduction of the applied voltage while achieving the desired expression may be an important improvement. One possible approach is to combine electrotransfer with exogenously applied heat. Previous work performed in vitro demonstrated that increasing temperature before pulsing can enhance gene expres sion and made it possible to reduce electric fields while maintaining expression levels. In the study reported here, this combination was evaluated in vivo using a novel electrode device designed with an inserted laser for application of heat. The results obtained in this study demonstrated that increased temperature during electrotransfer increased expression or maintained expression with a reduction in applied voltage. With further optimization this approach may provide the basis for both a novel method and a novel instrument that may greatly enhance translation of gene electrotransfer.

show abstract

“…Human MDR1 cDNA was subsequently transferred into human CD34 1 cells from cord blood or bone marrows and showed enrichment after chemoselection and improvement of chemotherapy tolerance [21À24], paving the way for human MDR1 clinical trials. MDR1 has also been introduced to keratinocyte stem cells by lentiviral vector for the selection of transduced cells and chemoprotection against topical application of colchicine in the treatment of skin disease [28]. A mutant form of P-glycoprotein (MDR1-F983A) was developed to protect hematopoietic cells from chemotherapeutic drugs in the presence of trans-(E)-flupentixol [25].…”

Section: Multidrug-resistant Gene (Mdr)mentioning

confidence: 99%