Current Pharmaceutical Biotechnology - Volume 2, Issue 1, 2001

Nonviral gene therapy has significant clinical potential, yet itstherapeutic utility has been hindered by low transfection efficiency due to acombination of extracellular and intracellular barriers. Recent developments informulation and delivery methodology have allowed a number of advances towardhigh efficiency gene delivery to various cell types and organs. In particular, theextracellular and intracellular pharmacokinetics of plasmid DNA trafficking are better understood in a number of cell systems. Using cationic lipid or polymers (often with receptortargeting), more than 10 5 plasmids can be delivered to a single cell. Endosomolytic agents promote endosomedisruption, and include weak bases, proton-sponge polymers, fusogenic peptides, viral particles, andphotosensitizing compounds. Both classical and nonclassical nuclear localization signal (NLS) peptides havealso been tested for enhancement of the probability of nuclear import events, a major rate-limiting step inDNA delivery to nondividing cells. For example, the M9 sequence from heterogeneous nuclearribonucleoprotein A1 (hnRNP A1) protein, a non-classical NLS, has been found to increase gene expressionlevel by more than 10 to 150-fold in a variety of cell types. This review will concentrate on the currentunderstandings of the basic mechanisms of nonviral gene delivery and new approaches in the field.

Recombinant Fv-immunotoxins are a new class of biologic anticanceragents composed of a recombinant antibody fragment linked to a very potentbacterial toxin. These potent molecules are designed to specifically bind and killcancer cells that express a specific target antigen on their cell surface. RecombinantFv-immunotoxins are an excellent example for the concept of rational drug design.They combine the progress in understanding cancer biology, -the recentknowledge on the mechanisms of malignant transformation and the specialproperties of cancer cells, -with the enormous developments in recombinant DNA technology and antibodyengineering. Recombinant Fv immunotoxins were developed for solid tumors and hematological malignancies and havebeen characterized intensively for their biological activity in vitro and in vivo in animal models. The excellentin vitro and in vivo activities of recombinant Fv-immunotoxins have lead to their pre-clinical developmentand to the initiation of clinical trial protocols.Recent trials have demonstrated potent clinical efficacy in patients with malignant diseases that are refractoryto traditional modalities of cancer treatment.It is thus suggested that this strategy can be developed into a separate modality of cancer treatment with thebasic rationale of specifically targeting cancer cells on the basis of their unique surface markers combined withpotent effective biological toxic agents that directly kill the cancer cell. Efforts are now being made to improvethe current molecules and to develop new agents with better clinical efficacy. In this review, we will describe the rationale in designing Fv-immunotoxins and will review current progress made in using these agents forcancer treatment.

Vaccination has been one of the most successful and cost-effective healthinterventions ever employed. One disease (smallpox) has been eradicated, another(poliomyelitis) should disappear early in the new millennium and a third (measles)should follow shortly after. Conventional vaccines usually depend on one of threedevelopment processes, attenuation of virulent organisms (by passage in cell cultureand(slash)or experimental animals), killing of virulent organisms (by chemical inactivation) or the purification of immunogenic molecules (either proteins or carbohydrates) from wholeorganisms. These traditional processes, although serendipitous and poorly understood, have produced effectivepharmaceutical products which give excellent protection against diseases such as smallpox, rabies, measles,yellow fever, tetanus and diphtheria. In spite of these successes however, the application of these protocolshave failed to produce safe and efficacious vaccines against other infectious diseases which kill or maim tens ofmillions of people every year. The most important of these are malaria, AIDS, herpes, dengue fever and someforms of viral hepatitis. Consequently, fundamentally new technologies are required to tackle these important infections. One of themost promising has been the development of genetically modified viruses. This process normally involvestaking a proven safe and efficacious vaccine virus, such as vaccinia or adenovirus, and modifying its genometo include genes coding for immunogenic proteins from other viruses such as HIV or measles. This reviewwill describe the generation of such novel vaccine vectors and compare their advantages and shortcomings. In addition the literature describing their use as experimental vaccines will also be reviewed.

Small heat shock proteins (sHSPs) belong to a family of 12- to 43-kDaproteins that are ubiquitous and are largely conserved in amino acid sequence among allorganisms. The principal heat-shock proteins that have chaperone activity (that is, they protectnewly made proteins from misfolding) belong to five conserved classes HSP100,HSP90, HSP70, HSP60 and the small heat-shock proteins (sHSPs). The sHSPs (which include alpha crystallin) can form large multimeric structures and have a wide range of cellular functions,including endowing cells with thermotolerance in vivo and being able to act as molecular chaperones in vitro sHSPs do this by forming stable complexes with folding -or unfolding- intermediates of their proteinsubstrates, probably the molten globule. This paper includes a brief survey of the chaperone family, the small heat shock protein superfamily,transcription of sHSPs, sequence comparisons and structural models of small heat shock proteins - structuralelements as potential drug targets, sHSPs as chaperone-like proteins, alpha crystallin chaperone-like activity,conformational diseases - the role of alpha crystallin small heat shock protein superfamily proteins, post-translationalmodification and useful pharmacological agents. Functionality of small heat shock proteins - targets and diseases where pharmacologically active agents are ofimportance, alpha crystallin- small heat shock proteins and prion diseases specific targets for diagnostic testsand drug development, details of some specific small heat shock proteins as drug targets, structural andfunctional implications for treatment.