Gene Therapy For Hyperlipidemia And Systemic Diseases

Hyperlipidemia, in both its acquired and inherited forms, has been clearly shown to be an independent risk factor for the development of atherosclerotic vascular disease, myocardial infarction, and stroke. The use of drug therapies in the treatment of acquired forms of this disease, through either 3-hy-droxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors or bile acid binding resins, has proven effective in decreasing the morbidity and mortality associated with hyper-lipidemia....

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McGee LH, Abel H, Hauswirth WW, Flannery JG. Glial cell line derived neurotrophic factor delays photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa. Mol Therapy 2001 4 622-629. Bok D, Yasumura D, Matthes MT, Ruiz A, Duncan JL, Chappelow AV, Zolutukhin S, Hauswirth W, LaVail MM. Effects of adeno-associated virus-vectored ciliary neuro-trophic factor on retinal structure and function in mice with a P216L rds peripherin mutation. Exp Eye Res 2002 74 719-735. Lewin AS, KA...

References

Vaccines W.B. Saunders, 1988. 2. Janeway CA, Travers P. Immunobiology. Current Biology Ltd. Garland 1994. 3. Stasney J, Cantarow A, Paschkis KE. Production of neoplasms by injection of fractions of mammalian neoplasms. Cancer Res 1955 11 775-782. 4. Paschkis KE, Cantarow A, Stasney J. Induction of neoplasms by injection of tumor chromatin. J. Natl. Cancer Inst 1955 15 1525-1532. 5. Ito Y. A tumor-producing factor extracted by phenol from papillomatous tissue of...

Gene Therapyspecific Safety Issues

As the development of gene therapy vectors for therapeutic gene delivery broadens and moves forward, the possibility of approved therapeutic products becomes more of a reality. However, regulatory approval and use of these therapeutic products in clinical trails requires a good safety profile. The use of gene therapy vectors carries with them certain safety issues that are specific to each vector type. Concerns specific to the safe use of gene therapy vectors include the generation of...

Advantages Of Using Molecular Adjuvants

The overall objective of any immunization strategy is to induce specific immune responses that could protect the immunized individual from a given pathogen over his or her lifetime. One major challenge in meeting this goal is that the correlates of protection from an individual pathogen vary from one infectious agent to the next. It would be a distinct advantage to design immunization strategies that can be ''targeted'' according to the correlates of protection known for the particular pathogen...

Biology Of Vaccinia

Poxviruses are classified into 2 subfamilies, chordopoxvirinae (vertebrate poxviruses) and entomopoxvirinae (insect pox viruses), and at least 46 species (11). The classification scheme is based on host range, sequence homology, and antigenicity. Vaccinia virus is a member of the orthopoxvirus genus. It is genetically distinct from both cowpox virus and variola virus (smallpox). All members of the orthopoxvirus genus have immune cross-reactivity and are genetically stable. This allowed for the...

Clinical Targets For Cancer Gene Therapy

Superficial transitional cell cancer of the bladder (TCC) is the fifth most common solid malignancy in the U.S. (229). Seventy percent to 80 of patients with bladder cancer present with these low-grade, noninvasive tumors confined to mucosa. Although most superficial cancers can be managed with periodic transurethral resection, this is not an ideal situation because recurrence is the rule (230). Even with close surveillance and follow-up, at least 50 will eventually require a cystec-tomy....

Bacteriamediated Dna Transfer Into Mammalian Cells

During infection, some bacteria have the capability to invade particular cells of the host. Dependent on their infection cycle, these microorganisms might establish themselves in different compartments of the target cell. Invasive bacteria such as S. typhimurium arrest in the phagosome, whereas facultative intracellular bacteria such as L. monocytogenes escape from the phagosome into the cytosol of the infected cell. Independent of whether the pathogens arrest in the phagocytic vacuole or in...

Status of Gene Therapy in the United States

As provided in the document, ''Application of Current Statutory Authorities to Human Somatic Cell Therapy Products and Gene Therapy Products,'' human gene therapy is defined as a medical intervention based on the administration of genetic material in order to modify or manipulate the expression of a gene product or to alter the biological properties of living cells. Cells may be modified ex vivo for subsequent administration or altered in vivo by gene therapy products given directly to the...

Neurodegenerative Diseases Amenable To Gene Therapy

The following discussion illustrates the most common forms of neurodegenerative diseases (summarized in Table 2) and potential gene therapy approaches that have been employed based on current understandings of disease mechanisms. Parkinson's disease (PD) is the second most common chronic neurodegenerative disease of humans. In 1995, the incidence ofPD was estimated to be between 1 100 to 1 500 individuals (17,18). This incidence translates to approximately 1 of the population over the age of 65...

Hematopoietic Systems And In The Skin

As noted above, studies on mice transgenic for human MDR1 established that constitutive overexpression of this gene protects animals from antineoplastic agents. Drugs could be administered safely at dose-levels several-fold higher than to mice of the respective background strains (10,11). To demonstrate the specificity of this protection, verapamil, an inhibitor of P-glycoprotein, was coadministered, resulting in reversal of drug resistance (12). Similarly, mice transgenic for a mutated...

Human Gene Transfer Trials

Hematopoietic Stem Cell Gene Transfer Studies The insertion of marker genes into hematopoietic stem cells has been useful in the evolution of gene transfer technology and has led to a better understanding of autologous stem cell transplantation. The first, and arguably the most successful of these studies was reported by Brenner et al. in 1993 (33). This study enrolled 20 patients under the age of 20 who were candidates for autologous bone marrow transplantation for acute myelogenous...

Properties Of Hematopoietic Stem Cells

The hematopoietic stem cell (HSC) is the ultimate progenitor of all of the cells found in the peripheral blood. In mammalian systems, small numbers of HSC have been shown to be capable of extensive proliferation, generating millions of mature blood cells in regulated numbers each day. HSCs are multipotent and differentiate into cells of the erythroid (red cell), megakaryocytic (platelets), myeloid (granulocytes and mono-cytes), and lymphoid (B- and T-cell) lineages (Fig. 1). HSC can self-renew...

Therapeutic Applications Of Transcleaving And Transsplicing Ribozymes

Ribozymes have the potential to become useful therapeutic agents, and currently they are being developed for a wide variety of clinical applications. The vast majority of effort has been expended in the development of trans-cleaving hammerhead and hairpin ribozymes as inhibitors of viral gene expression. In particular, these ribozymes have been targeted to cleave and destroy HIV-1 RNAs to inhibit viral replication in infected cells. The effectiveness of ribozymes as inhibitors of cellular gene...

Extracellular Barriers For Gene Delivery

For in vivo applications, the development of polyplexes is not only confronted with intracellular barriers, but also with extra cellular barriers, which are those prior to reaching the target cells. A schematic diagram of the potential intracellular and extracellular fate of a polyplex is shown in (Fig. 3). Strategies have to be developed in designing polyplexes that enable them to survive blood and other biological fluids, and to escape extracellular physical barriers, in order to reach the...

Improvements in Vector Design

The basic arrangement described above is functional but unsatisfactory in several ways. In particular, the considerable sequence overlap that exists between the vector and packaging components means that there is a very high risk of recombination occurring that could create an infectious replication-competent retrovirus (RCR) (8). Overlap occurs because extensive sequences of the gag gene are retained in the vector to enhance the efficiency of packaging (9), although Gag protein expression...

Pnp

Viral thymidine kinases and a poor substrate for mammalian thymidine kinases, concentrations can be achieved that are lethal to cells expressing HSV-tk but are nontoxic to normal mammalian cells (10,11,15). Culver et al. demonstrated the first in vivo application of suicide gene therapy for cancer using retroviral-mediated HSV-tk gene transfer via fibroblast packaging cells injected into brain tumors in mice (16,17). Since this initial study, HSV-tk suicide gene therapy has been applied and...

Mlv

Figure 1 Diagrams drawn from cryoelectron micrographs of cross-sections through vitrified films of various types of liposomes and DNA-liposome complexes. SUVs condense nucleic acids on the surface and produce ''spaghetti and meatballs'' structures. MLVs appear as ''Swiss rolls'' after mixing with DNA. BIVs are produced using a formulation developed in our laboratory (14). Nucleic acids are efficiently encapsulated between 2 BIVs. Figure 1 Diagrams drawn from cryoelectron micrographs of...

Pharmacokinetics Of Oligonucleotides

Cellular uptake of phosphorothioate oligonucleotides has been documented to occur in most mammalian cells (96-103). Cellular uptake of oligonucleotide is time and temperature dependent. It is also influenced by cell type, cell culture conditions and media, and the length sequence of the oligonucleotide itself (96). No obvious correlation between the lineage of cell, whether the cells are transformed or virally infected, and uptake has been identified. Cellular uptake appears to be an active...

Tengisis Disease

Airenne Department of Molecular Medicine, AI Virtanen Institute, University of Kuopio, and Ark Therapeutics Oy, Kuopio, Finland Kurosh Ameri Cancer Research UK, Molecular Oncology Laboratories, Weatheral Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom Helen A. Andersson Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, U.S.A. W. French Anderson Norris Cancer Center, University of Southern California Keck School of Medicine, Los...

In Vivo Models

The ability of HSC to maintain lifelong hematopoiesis can only be assayed in vivo. Several animal models have been developed for assaying both murine and human HSC activity in vivo. W W mice have a spontaneous mutation in their c-kit receptor and die shortly after birth due to anemia and cytopenia unless rescued by transplantation with normal WBM (90). Thus, the systemic deleterious effects of lethal irradiation are avoided by using these mice as an in vivo model. W W mice have been used to...

Candidate Diseases For Cutaneous Gene Therapy

Many skin diseases are characterized by single gene mutations, either dominant or recessive, and can therefore possibly be corrected by destruction of the mutant gene product or introduction of a wild-type gene product. In addition to being used to correct inherited skin diseases, the skin can be used as a delivery system to secrete various polypeptides, such as enzymes, growth factors, and cytokines, into the systemic circulation. Furthermore, taking advantage of the potent antigen-presenting...

Viral Replication

The Ad viral life cycle is understood best for subgroup C, which is another factor in the choice of Ad5 and 2 as gene therapy vectors (Fig. 3). The knob of the fiber protein binds to the CAR receptor (13), followed by an interaction of the RGD sequence in the penton base with cell surface aVp3 or aVp5 integrins (15). Excess soluble integrins inhibit Ad internalization but not binding, suggesting that penton base-integrin interaction is instrumental in internalization (16). Ad modified with...

Tissue Specific Expression

Since the initial report by Aihara and Miyazaki (51), in vivo electroporation of the skeletal muscle for delivery of therapeutic proteins has become widely used. Table 2 summarizes some of the recent literature reports in which electroporation was used to enhance plasmid delivery to skeletal muscle. Although the devices, conditions, methods, and animal models substantially differ, all studies conclude that plasmid injection followed by electroporation can be successfully used to deliver...

In Vitro Studies

The methods outlined above have been used to make a large number of first-generation E1, E3 adenoviral vectors. Among the most widely used are those that express readily monitored reporter genes such as p-galactosidase, luciferase, chloramphenicol acetyl transferase, and green fluorescent protein (34,35). As a control, viruses with the same promoter-driving expression of no transgene (AdNull) are used. Using the reporter gene Ad vectors, there are many studies examining the ease of gene...

Ecdysoneresponsive Regulatory System

The steroid hormone ecdysone, which is normally found in mammals, plays a fundamental role during Drosophila molting and metamorphosis. Pulses of 20-hydroxyecdysone mediate that cascade of morphological modification, which results in degeneration of larval tissues and development of adult structures (162). Ecdysone acts by stimulating a transcription factor, the ecdysone receptor (EcR). The EcR is a member of the nuclear receptor superfamily, and its modular organization resembles that of...

Brief History Of Biolistics And Gene Guns

Biolistic technology was originally developed in the area of plant genetics to introduce genes into target cells that were largely refractory to traditional methods of gene delivery (1). Biolistic lore holds that the gene gun was inspired by interactions between edible bonsai plants and squirrels, and the later involvement of an air rifle to control these interactions. Legend has it that in the course of those events, Dr. John Sanford made the pivotal extrapolation that if an air rifle could be...

Host Responses And Toxicity

Host immune responses, including innate immune responses, cellular immunity, or humoral antibody responses, may hinder the use of some gene therapy vectors. However, administration of AAV vectors has not been reported to induce innate immune responses or proinflammatory cytokines. Also, AAV vectors are replication defective and contain no viral genes, so cellular immune responses against the viral components should not be evoked readily. In all the in vivo studies of AAV vectors in rodents,...

Overcoming Barriers To Airway Gene Transfer

In the preceding section of this chapter, the apical membrane of WD airway cells has been established as a formidable barrier to efficient transduction by all the current gene transfer vectors. Intravenous approaches might overcome many of the apical membrane barriers (123,124), but the multiple barriers that must be crossed (e.g. endothelium, endothelial basement membrane, interstitium, epithelial basement membrane), combined with the increased possibility of systemic toxicity inherent in...

Dna Vaccines

Recent work from a number of laboratories has demonstrated that the injection of a DNA plasmid containing foreign genes for proteins of a pathogen or cancer antigens directly into a host results in the subsequent expression of the foreign gene in that host and the presentation of the specific encoded proteins to the immune system (Fig. 1). DNA vaccine constructs are produced as small circular vehicles or plasmids. These plasmids are constructed with a promoter site that starts the transcription...

Transduction of the Liver

The liver is very attractive target for gene therapy. The fenes-trated structure of its endothelium permits exposure of the parenchymal cells to intravenously delivered vector and secretion of vector-encoded therapeutic proteins into the circulation for systemic delivery. Ads are particularly attractive vectors for liver-directed gene therapy because of their efficiency at transducing hepatocytes following intravenous injection. -Antitrypsin antagonizes neutrophilic elastase and is abundantly...

Monitoring Gene Therapy Levels

Precise localization and quantitative assessment of the magnitude and temporal variation of transgene expression is a necessary component of any gene therapy trial. Direct imaging with a transgene-specific imaging probe is ideal but neither feasible nor practical in most cases. To develop a specific probe for each individual transgene is not always technically possible furthermore, it is necessarily labor and cost-intensive. Using Figure 13 Tracking HSV Infection with 131I FIAU Using...

Development Of Ex Vivo Gene Delivery By Myoblast Transplantation

Evidence for Myogenic Precursor Cells Myoblasts Skeletal muscle comprises approximately 10 of the total human body mass and is highly accessible to manipulation. A typical striated mature skeletal muscle cell, known as a myofiber, is large (1-40 mm in length and 10-50 microns wide), cylindrical, and multinucleated (as many as 100 nuclei per cell). Myofibers contain many nuclei because they are formed during development by the fusion of mononucleated precursor cells known as myoblasts....

Intracellular Trafficking And Barriers

Once the cell has been targeted and the DNA has been internalized via receptor-mediated endocytosis, several intracellular barriers (Fig. 2) need to then be overcome for successful foreign gene expression to occur. To tackle the intracellular barriers, a polyplex needs to Survive the cytoplasmic environment Traffick the cytoplasmic environment targeting the nucleus with subsequent nuclear entry Be disassembled in the nucleus so it can be recognized by the cell's transcription machinery and be...

Familial Hypercholesterolemia

Synthetic vectors are being evaluated for the treatment of familial hypercholesterolemia (FH) and hyperlipidemias. FH is an autosomal dominant disorder caused by mutations in the low-density lipoprotein (LDL) receptor. Patients have persistently elevated levels of LDL in their serum, which leads to the development of atherosclerosis and coronary artery disease (43). Tomita et al. (44) injected a complex of hemagglutinat-ing virus of Japan (HVJ) liposomes and a plasmid expressing the human LDL...

Gene Therapy Of Specific Disorders

A comprehensive review of the tremendous progress in the gene therapy for IEM is beyond the scope of this review. In fact, gene therapy studies have been conducted in almost every type of IEM. Instead, specific IEM were chosen either because they illustrate the above principles or for their important historical role. The aminoacidopathies are a heterogeneous group of reces-sively inherited enzyme deficiencies that are associated with the accumulation of specific amino acids in blood and other...

Reporter Genes And Reporter Probes

Reporter Cell

There are essentially 4 different types of optical reporter genes currently in use for studies in living animals. These genes either encode a) a protein that contains a chromophore (a short, internal peptide segment that contributes to the protein's fluorescent capabilities following obligatory posttranslational modifications), which fluoresces when externally irradiated (fluorescence imaging), b) an enzyme that can convert an ex-ogenously added, optically quiescent substrate into a fluorescent...

Vector Transgene Expression

We have explored the activity of many viral and cellular promoters in the background of replication-defective viral vectors (47,110-118). These lytic gene promoters display transient activity in both neuronal and nonneuronal cells. Thus, viral IE promoters are effective for applications that require only transient transgene expression. The human cytomegalovirus (HCMV) IE gene promoter produced vigorous transgene expression for up to 21 days post infection in vivo (Ramakrish-nan and Fink,...

Antisense Oligonucleotide Chemistry

Antisense Oligonucleotides

The most advanced oligonucleotide chemistry used for antisense drugs is phosphorothioate oligodeoxynucleotides. These differ from natural DNA in that 1 of the nonbridging oxygen atoms in phosphodiester linkage is substituted with sulfur (Fig. 1). Phosphorothioate oligodeoxynucleotides are commercially available, easily synthesized, support RNase H activity, exhibit acceptable pharmacokinetics for systemic and local delivery, and have not exhibited major toxicities that would prevent their use...

Basis For Gene Therapy For

One common type of IEM is caused by deficiency of an enzyme that catalyzes the conversion of one chemical to another (Fig. 2a). Deficiency of a specific enzyme can cause disease through 3 separate mechanisms (1) excessive accumulation of substrate to toxic levels, (2) deficiency of an essential product, or (3) metabolism of the substrate through alternative biochemical pathways leading to toxic secondary metabolites. Examples of such IEM include phenylketonuria and meth-ylmalonic aciduria. IEM...

Considerations for the Application of Ex Vivo Gene Therapy to AD

Rodent and primate studies in cholinergic denervation indicate that NGF is important in rescuing cells from axotomy-induced cell death as well as potentially augmenting cholinergic function. Therefore, to evaluate the effectiveness of NGF as a therapy for AD, patients to be treated would ideally be in the early stages of the disease, so that the rate of disease progression can be assessed. Outcomes should be assessed with careful preoperative and postoperative monitoring with standard...

Molecular Forces

The Origin and Measurement of Molecular Forces We divide these forces into two broad categories, both of which can be either attractive or repulsive. First, there are interactions that are connected with fields emanating from sources within or on the macromolecules themselves (16) (e.g., electrostatic fields pointing from the fixed-charge distributions on macromolecules into the surrounding space, fields of connectivity of hydrogen bond networks extending from the macromolecular surfaces...

Engineering Mdr Vectors To Improve Efficiency Of Drug Selection

One of the goals of gene therapy is to modify cells genetically such that they can supply a useful or necessary function to the cell (3). One of the most promising applications of the MDR1 gene in therapeutic vectors as a selectable marker in vivo is the protection of bone marrow cells during intensive chemotherapy. During chemotherapy, the MDR1 gene is transduced or transfected into drug-sensitive bone marrow cells and selected for by exposure to MDR agents. The untransfected untransduced...

Oligonucleotide Formulations

Physical-chemical Properties Due to the presence of a mixture of diastereisomers, phospho-rothioate oligodeoxynucleotides are amorphous solids possessing the expected physical properties of hygroscopicity, low-bulk density, electrostatic charge pick up, and poorly defined melting point prior to decomposition. Their good chemical stability allows storage in the form of a lyophilized powder, spray-dried powder or a concentrated, sterile solution more than 3 years of storage is possible at...

A HSV Gene Transfer for Neuropathy and Pain

Gastrocnemius Muscle Transfer

In some sense, the most natural target for the therapeutic use of HSV-mediated gene transfer is the PNS. Recombinant rep lication-defective vectors, such as wild-type HSV, target with high efficiency to dorsal root ganglion DRG neurons following subcutaneous inoculation, a process that benefits from high-level expression of the HveC HSV receptor on sensory nerve terminals in the skin 160 and specific interactions between capsid and tegument proteins and dynein to mediate retrograde axonal...

Gene Therapy for Childhood Onset Blindness

University of Pennsylvania Philadelphia, Pennsylvania, U.S.A. I. BACKGROUND GENE THERAPY AND LEBER CONGENITAL AMAUROSIS In recent years, mutations in a large number of different genes have been implicated in the pathophysiology of retinal degenerative diseases RetNet, http www.sph.uth.tmc.edu Re-tNet . Among these are mutations that lead to particularly severe early onset blinding disease. With the recent developments of methods for in vivo retinal gene delivery reviewed by 5,7,12,15 , it has...

Cardiovascular Trials Of Fgf Protein And Gene Therapy

Several investigators have shown improvements in muscle perfusion in animal models of hindlimb ischemia using recombinant FGF 84-86 . The safety of intra-arterial bFGF administration in patients with intermittent claudication was recently demonstrated 87 . In this phase I, double-blind, placebo-controlled clinical trial there was improvement in calf blood flow by strain gauge plethysmography in bFGF-treated patients at 6 months compared with controls. To date, the TRAFFIC study is the only...

Bicistronic Vectors Containing Selectable Markers

Although coexpression of two proteins can be achieved through the use of separate promoters, the coexpression is frequently uncoupled due to promoter interference or shutoff of gene expression from one of the promoters, which causes the selected cells not to express the desired protein. To overcome this problem, the selectable marker may be expressed with the therapeutic gene as a translational or transcriptional fusion. A therapeutic protein can be directly linked to the carboxylterminus of...

Replicationcompetent or Wildtype AAV

Wild-type AAV is not a human pathogen but generation of wild-type or rcAAV during vector production needs to be avoided for several reasons. The presence of wild-type AAV in vector preparations may increase the likelihood of vector mobilization following a helper virus infection in the patient, which could increase the likelihood of cellular immune responses to AAV proteins and can cause significant alterations in the biology of the vector because of the pleiotropic effects of the rep proteins....