Peptide delivery to tissues via reversibly linked protein transduction sequences.

The development of peptide-based therapeutics has suffered from challenges associated with delivery to intact tissue. In skin, an array of protein targets resides only tens of micrometers below the surface; however, because of difficulties in traversing the cutaneous barrier, the potentialfor peptide-based therapeutics remains unrealized. We have developed a general approach for topical peptide delivery into skin using releasable protein transduction sequences to enable peptide transport across tissue boundaries. Upon entry into the cell, the disulfide linkage between the peptide transduction sequences and peptide cargo is cleaved, permitting the dissociation of the highly charged peptide transduction sequences from the active peptide. A protype cargo peptide, the hemagglutinin (HA) epitope, was conjugated to a hepta-arginine protein transduction sequence via a releasable disulfide linkage. This construct penetrated the skin to deep dermis within 1 h after topical application. Consistent with the dissociation of the protein transduction and cargo sequences, absorbed protein transduction sequences and HA peptides displayed differential intracellular localization. Reversible protein transduction sequence linkage thus represents a noninvasive platform for tissue delivery of intact peptides with no requirement for viral vectors or parenteral injection and may be of broad utility in molecular therapy.

Impact of laminin 5 beta3 gene versus protein replacement on gene expression patterns in junctional epidermolysis bullosa.

Molecular therapy studies to date have examined only a limited number of corrective parameters. To assess more global impacts on cellular gene expression for two major molecular therapeutic approaches, we compared gene versus protein delivery in the human genetic disease junctional epidermolysis bullosa (JEB). Both gene and protein replacement of the laminin 5 beta3 (beta3) adhesion molecule restored normal growth and adhesion to poorly viable JEB cells. Gene expression profiling was then performed using cDNA microarrays. The expression of more genes was normalized after beta3 gene transfer than after protein transfer. As anticipated for beta3 delivery, many of the genes whose expression was restored to the normal range were those encoding adhesion molecules and hemidesmosome components. Although gene transfer normalized the expression of a higher percentage of genes than did protein transfer, neither approach fully normalized expression of all genes examined. In addition, both approaches disrupted the expression of some genes, but protein transfer altered expression of a larger proportion of the genes studied. Our findings suggest that therapeutic gene and protein delivery may exert different effects on gene expression and thus may have implications for the development and analysis of molecular therapies for the treatment of genetic disorders.

In vivo restoration of laminin 5 beta 3 expression and function in junctional epidermolysis bullosa.

The blistering disorder, lethal junctional epidermolysis bullosa (JEB), can result from mutations in the LAMB3 gene, which encodes laminin 5 beta3 (beta3). Appropriate expression of LAMbeta3 in JEB skin tissue could potentially ameliorate the symptoms of the underlying disease. To explore the utility of this therapeutic approach, primary keratinocytes from six unrelated JEB patients were transduced with a retroviral vector encoding beta3 and used to regenerate human skin on severe combined immunodeficient (SCID) mice. Tissue regenerated from beta3-transduced JEB keratinocytes produced phenotypically normal skin characterized by sustained beta3 expression and the formation of hemidesmosomes. Additionally, beta3 gene transfer corrected the distribution of a number of important basement membrane zone proteins including BPAG2, integrins beta4/beta1, and laminins alpha3/gamma2. Skin produced from beta3-negative (beta3[-]) JEB cells mimicked the hallmarks of the disease state and did not exhibit any of the aforementioned traits. Therefore, by effecting therapeutic gene transfer to beta3-deficient primary keratinocytes, it is possible to produce healthy, normal skin tissue in vivo. These data support the utility of gene therapy for JEB and highlight the potential for gene delivery in the treatment of human genetic skin disease.

Baker's cysts mimicking the symptoms of deep vein thrombosis: diagnosis with venous duplex scanning.

PURPOSE: The purpose of this study was to determine the incidence and characteristics of Baker's cysts discovered during venous duplex examinations to rule out deep vein thrombosis (DVT). METHODS: We reviewed the vascular laboratory charts of patients found to have Baker's cysts during venous duplex studies to rule out DVT from October 1988 through December 1995. RESULTS: Ninety-five (3.1%) of 3072 patients who underwent venous duplex studies were found to have 111 Baker's cysts. Seven of the 95 had coexistent DVT. Ten patients had ruptured cysts, whereas six patients had cysts that compressed the popliteal vein. CONCLUSION: The presentation of DVT and that of a Baker's cyst are similar enough to be difficult to distinguish by clinical examination. Careful examination of the popliteal fossa should be performed during venous duplex examinations regardless of the indication for the study.

The effect of dimethyl sulfoxide on gray matter injury in experimental spinal cord trauma.

The possible effect of dimethyl sulfoxide upon the development of lesions in the gray matter after experimental spinal cord trauma has been investigated with the use of cytochrome oxidase assay and quantitative histologic measurement of total liquefaction necrosis. Observations were made in 17 unconditioned dogs receiving an impact trauma of 400 gm cm force. Experimental animals were given 2.5 gm/kg of dimethyl sulfoxide in 40% solution intravenously one hour prior to trauma, and control animals received a similar volume of saline. No reduction could be found in the degree of loss of cytochrome oxidase at one hour after trauma, nor in the extent of acute necrosis. A slight but non-significant increase in the amount of hemorrhage was noted in gray matter at the trauma site following treatment with dimethyl sulfoxide. The agent resulted in an increase in cytochrome oxidase activities in nontraumatized control gray matter.

Vasoactive intestinal polypeptide in tissues of the human female reproductive tract.

Vasoactive intestinal peptide (VIP) is a known potent hypotensive and vasodilatory agent. Studies were performed to determine the possible role of VIP in the human menstrual cycle in 44 patients in the reproductive age group, some of whom were using oral contraceptives, three pregnant patients, and four postmenopausal patients. No significant relationship between VIP and menstrual cycle phase was found.