Medical brain notes 37

  Gene Therapy INTRODUCTION The pioneering report of James Watson and Francis Crick describing the helical structure of DNA spurned an upsurge of biomedical research focusing on the composition of DNA, RNA and proteins and their role in health and disease that continues today. This “molecular revolution” has markedly influenced understanding of the pathophysiology of a diverse collection of disease states ranging from cystic fibrosis (CF), inborn errors of metabolism and immunodeficiencies to cancer, cardiovascular disease and diabetes. Rapid development of recombinant DNA technology prompted sequencing of the human genome and identifying genotype-phenotype rela-tionships in human disease. Although these efforts have produced highly sophisticated, extremely sensitive diagnostic tests, the development of successful molecular therapies based upon this expanded knowledge of disease pathogenesis is still in progress. Gene therapy is the use of nucleic acids as therapeutic medicinal com...

  EX VIVO VERSUS IN VIVO GENE THERAPY Gene therapy is a means of treatment for diseases that have limited or no therapeutic options. Once a disease has been identified as a possible candidate, the gene necessary for treatment must be identified and cloned. Enough must be understood about the disease and the gene product in order to ensure that the therapeutic component is delivered to the appropriate cellular compartment responsible for its processing and sub-sequent biological activity. The necessity for long-term gene expression or requirements that expression be timed with other biological processes will influence the design of the vector component of the therapy. The target tissue/organ must be readily accessible and a defined, measurable endpoint must be identified for assessment of therapeutic efficacy. Several strategies can be used for gene transfer. Direct injection of vector/ DNA complexes into the bloodstream is often char-acterized by low levels of gene expression, maki...

  GENE THERAPY IN THE CLINIC: DISEASE TARGETS There are currently 1,260 active gene therapy clinical trials worldwide (Anonymous, 2006). Approximately 67% of these trials are for cancer. Treatment of monogenetic diseases, the premise of early gene transfer experiments, is the goal of only  ~ 8% of active clinical trials. General indications for all gene therapy trials in the clinic are summarized in Table 1. Currently, gene therapy trials are primarily held in the United States (65% of all trials), the United Kingdom (12%) and Germany (5.9%). The geographical distribution of gene therapy clinical trials is summarized in Table 2. Gene Therapy for Cancer   The objective of cancer gene therapy is to destroy tumor cells and preserve normal tissue. Strategies to achieve this goal include: ( a ) correction of genetic mutations contributing to the malignant phenotype, ( b ) stimulation of a T-cell-mediated immune response against the tumor (immunotherapy), ( c ) use of oncolytic...

  VECTORS FOR GENE TRANSFER General Considerations   Preparations for gene delivery are colloidal suspen-sions consisting of complex molecules with average hydrodynamic radii ranging from 40 to 1,000 nm. Particles of this size are readily taken up by organs of the reticuloendothelial system (RES) such as the liver, spleen, bone marrow and adrenal glands and effi-ciently cleared from the circulation. They are also highly susceptible to opsonization, where they are coated with serum proteins such as complement and taken up by macrophages in the liver and spleen within minutes after intravenous administration (Dash, 1999; Read, 2005). Conjugation of vectors with biodegradable polymers has been shown to success-fully prevent interaction with the RES and serum components (Oupicky, 2002; Demeneix, 2004; Kommareddy, 2005), however, this effect is often avoided by instillation of the vector directly into the target organ or tissue. Even though a vector is administered by direct inject...

  General Anatomy and Production of a Gene Transfer Vector A gene-based medicine typically consists of an expression cassette made of cDNA flanked by apromoter on the 5 0  side and a transcription stop and polyadenylation site on the 3 0  side (Fig. 4A). This is incorporated into a DNA plasmid or a recombinant virus, based upon therapeutic requirements (Tables 4 and 5). The genes responsible for the pathogenicity of viral vectors are removed and replaced with the expression cassette in order to limit virus reproduc-tion and fulminant disease. In many vectors, all that remains of the original virus genome are long terminal repeats (LTRs), 5 and 3 terminal regions of the virus that control transcription for RNA viruses or inverted terminal repeats (ITRs), identical but oppo-sitely oriented sequences that drive DNA replication and stabilize the genome of DNA viruses. The packaging signal ( y ), responsible for virus assembly, is also kept intact. Genes for replication are su...

  Viral Vectors for Gene Transfer   Viruses, natural parasites that efficiently enter cellular targets and hijack cellular machinery for propagation, are currently the most effective vectors for gene therapy. Approximately 70% of all gene therapy clinical trials employ viral vectors (Table 4). Many viruses with divergent properties have been devel-oped for gene transfer. Retroviruses, adenoviruses and adeno-associated viruses (AAVs) are the most extensively studied to date. Characteristics of each are summarized in Table 5. Retroviral Vectors Biology   Retroviruses are 80 to 100 nm in diameter and contain two copies of a single-stranded RNA genome, 7 to 11 killobases (kb) in length. Within the capsid, the RNA is in close association with reverse transcriptase (RT), integrase (IN), and protease (PR) enzymes, necessary for virus replication (Fig. 5A). These elements are surrounded by a shell of nucleocapsid (NC) proteins enclosed in the capsid matrix (MA), which forms the c...

  Viral Vectors for Gene Transfer   Viruses, natural parasites that efficiently enter cellular targets and hijack cellular machinery for propagation, are currently the most effective vectors for gene therapy. Approximately 70% of all gene therapy clinical trials employ viral vectors (Table 4). Many viruses with divergent properties have been devel-oped for gene transfer. Retroviruses, adenoviruses and adeno-associated viruses (AAVs) are the most extensively studied to date. Characteristics of each are summarized in Table 5.   Adenoviral Vectors Biology   Adenoviruses are non-enveloped, lytic, DNA viruses with a linear double-stranded genome and icosahedral symmetry. Since the isolation of the first human adenovirus in 1953 from tonsils and adenoid tissue (Rowe, 1953), 50 additional serotypes have been identified and grouped into six species (A–F) based on genome size, composition and homology, hemag-glutinating properties and oncogenicity in rodents. Because the subg...