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PubMed2005

Barriers to gene delivery using synthetic vectors.

Progress has been made in the development of different types of nucleic acids such as DNA and siRNA with the potential to form the basis of new treatments for genetic and acquired disorders. The lack of suitable vectors for the delivery of nucleic acids, however, represents a major hurdle to their continued development and therapeutic application. Synthetic vectors based on polycations are promising vectors for gene delivery as they are relatively safe and can be modified by the incorporation of ligands for targeting to specific cell types. However, the levels of gene expression mediated by synthetic vectors are low compared to viral vectors. The aim of this chapter is to give an overview of the main barriers that have been identified as limiting gene transfer using polycation-based synthetic vectors. The chapter is divided into two sections to focus on both extracellular and intracellular barriers. We describe novel strategies that are being used to develop increasingly sophisticated vectors in an attempt to overcome these barriers. For instance, we describe approaches to prolong the plasma circulation of polyplexes by the incorporation onto their surface of hydrophilic polymers such as polyethylene glycol (PEG) and poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). In addition, strategies to improve transfer of nucleic acids from the outside of the cell to the nucleus are described to overcome barriers such as escape from endocytic vesicles and translocation across the nuclear membrane. Furthermore, we highlight new types of vectors based on reducible polycations that are triggered by the intracellular environment to facilitate efficient cytoplasmic release of nucleic acids.

Active Transport, Cell Nucleus↗
PubMed2005

[The effects of brain-derived neurotrophic factor gene-modified neural stem cells on primary cultured dorsal root ganglion cells in vitro].

OBJECTIVE: To gain stable genetic modification of neural stem cells (NSC) that constitutively secrete brain-derived neurotropic factor (BDNF) and to explore the biological role on the survival and neurite outgrowth of cultured dorsal root ganglion (DRG) neurons. METHODS: BDNF gene fragment from human brain cDNA libraries was obtained by using PCR. With molecular cloning technique, the recombinant stem cell viral vector with report gene was constructed, which is that MSCV-BDNF-IRES(2)-EGFP vector could encode Polycistronic mRNA. Viral particle was packaged by PT67 cell line and infected neural stem cells (mouse clone: C17.2). After selection with cloning cylinder, the expression of BDNF was assessed by immunohistochemistry enzyme linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR). The effects of stable gene-modified neural stem cells on embryonic mouse DRG neurons were evaluated in a dual-chambered cocultivation system at 3th, 10th day. RESULTS: RT-PCR analysis demonstrated expression of mRNA for human-BDNF. ELISA confirmed the presence of secreted BDNF 24 h after transfection and showed that the level of BDNF production by NSC-BDNF transfected C17.2 was at a rate of (14.6 +/- 0.8) ng x d(-1) x 10(-6) cells even after 3 months. With immunohistochemical analysis, compared with the control, the longer neurite outgrowth of cultured DRG cells and the more survival neurons were observed in NSC-BDNF transfected cells group. CONCLUSIONS: BDNF gene could be stably expressed in C17.2 cell line by MSCV, and the BDNF gene-modified NSC markedly enhances the survival and neurite outgrowth of cultured DRG neurons.

Animals↗
PubMed1997

Vectors--shuttle vehicles for gene therapy.

Gene therapy is being considered for the treatment of various inherited and acquired disorders. The basic premise of this new therapeutic modality is manipulation of gene expression towards a therapeutic end. The early development of the field focused on a technique called ex vivo gene therapy in which autologous cells are genetically manipulated in culture prior to transplantation. Recent advances have stimulated the development of in vivo gene therapy approaches based on direct delivery of the therapeutic gene to cells in vivo. The rate-limiting technologies of gene therapy are the gene delivery vehicles, called vectors, used to accomplish gene transfer. The most efficient vectors are based on recombinant versions of viruses with retroviral vectors serving as prototypes. This viral vector system has been exploited in ex vivo approaches of gene therapy in which cultured, dividing cells are transduced with the recombinant virus resulting in integration of the proviral DNA into the chromosomal DNA of the recipient cell. The use of retroviral vectors in gene therapy has been restricted to ex vivo approaches because of difficulties in purifying the virion and the requirement that the target cell is dividing at the time of transduction. More recently, vectors based on adenoviruses have been developed for in vivo gene therapy. These viruses can be grown in large quantities and highly purified. Importantly, they efficiently transduce the recombinant genome into non-dividing cells. Applications include in vivo gene delivery to a variety of targets such as muscle, lung, liver and the central nervous system. Clinical trials of in vivo delivery with adenoviruses have been undertaken for the treatment of cystic fibrosis.

Cystic Fibrosis↗
PubMed2004

The clinical potential of antiepileptic gene therapy.

Epilepsy afflicts approximately 1% of the population and, although the majority of patients gain effective seizure control through existing medications, a significant number prove refractory to treatment. For intractable focal epilepsies, gene therapy techniques provide a realistic treatment alternative, especially in patients who are considered surgical candidates. Neurotransmitter receptors and ion channels offer attractive gene therapy targets, but the pattern of viral vector transduction and gene expression can dramatically influence the final outcome. Recently, studies have shown that viral vector-mediated transduction and expression of neuroactive peptides, such as galanin and neuropeptide Y, can attenuate seizure sensitivity and prevent seizure-induced cell death in vivo. As future studies define the best means to avoid immunological silencing, as well as establish transduction properties in pathological, epileptic tissue, it should be possible to develop an efficacious gene therapy for intractable focal epilepsy.

Animals↗
PubMed1996

In vivo expression of full-length human dystrophin from adenoviral vectors deleted of all viral genes.

Adenoviral vectors have been shown to effect efficient somatic gene transfer in skeletal muscle and thus offer potential for the development of therapy for Duchenne muscular dystrophy (DMD). Efficient transfer of recombinant genes has been demonstrated in skeletal muscle using recombinant adenoviruses deleted of E1. Application of this vector system to the treatment of DMD is limited by the vector immunogenicity, as well as by size constraints for insertion of recombinant genes, precluding the incorporation of a full-length dystrophin minigene construct. We describe in this study the use of helper adenovirus to generate a recombinant vector deleted of all viral open reading frames and containing a full-length dystrophin minigene. We show that this deleted vector (delta vector) is capable of efficiently transducing dystrophin in mdx mice, in myotubes in vitro and muscle fibers in vivo. Our modification of adenoviral vector technology may be useful for the development of gene therapies for DMD and other diseases.

Adenoviridae↗
PubMed2006

Prevention of cytotoxic T lymphocyte responses to factor IX-expressing hepatocytes by gene transfer-induced regulatory T cells.

Treatment of genetic disease such as the bleeding disorder hemophilia B [deficiency in blood coagulation factor IX (F.IX)] by gene replacement therapy is hampered by the risk of immune responses to the therapeutic gene product and to the gene transfer vector. Immune competent mice of two different strains were tolerized to human F.IX by hepatic gene transfer mediated by adenoassociated viral vector. These animals were subsequently challenged by systemic administration of an E1/E3-deleted adenoviral vector, which is known to induce a cytotoxic T lymphocyte response to the transgene product. Immune tolerance prevented cytotoxic T lymphocyte activation to F.IX and CD8(+) cellular infiltrates in the liver. Moreover, a sustained and substantial increase in hepatic F.IX expression from the adenoviral vector was achieved despite in vitro T cell responses to adenoviral antigens. Cytolytic responses to therapeutic and to viral vector-derived antigens had been prevented in vivo by activation of regulatory CD4(+) T cells, which mediated suppression of inflammatory lymphocyte responses to the liver. This result suggests that augmentation of regulatory T cell activation should provide new means to avoid destructive immune responses in gene transfer.

Adenoviridae↗
PubMed1998

Ribozyme gene therapy for hepatitis C virus infection.

BACKGROUND: The development of antiviral drugs for hepatitis C virus (HCV) infection represents a substantial challenge. Similar to human immunodeficiency virus (HIV), HCV is highly prone to mutation. It is, therefore, expected that potential HCV therapeutics currently under development, such as protease inhibitors, will suffer from the same shortcomings of HIV therapeutic drugs; the emergence of drug resistant viral mutants. Ribozymes (Rz) are enzymatic RNA molecules that can be engineered to specifically target any given RNA molecule. A therapeutic Rz can be manufactured and administered as a drug, or a Rz gene can be delivered and expressed intracellularly by gene therapy. For HCV therapeutics, we favour the gene therapy approach as delivery and in vivo expression of Rz genes will result in a constant and continuous supply of multiple intracellular Rz, offering less opportunity for the development of drug-resistant viral variants. OBJECTIVES: To utilise direct intravenous injection of hepatotropic viral vectors to transfer Rz genes directly into the hepatocytes of HCV-infected patients, resulting in degradation of the HCV positive strand RNA genome, the viral mRNAs, and even the negative strand RNA replication intermediate. We plan to circumvent the emergence of drug-resistant viral mutants by targeting multiple, highly conserved HCV RNA sequences simultaneously with multiple Rz genes expressed from a single vector. STUDY DESIGN: Rzs targeting conserved regions of the HCV positive and negative RNAs were transcribed in vitro and used to cleave HCV target RNAs. The most effective Rzs identified were then incorporated into adeno associated viral (AAV) vectors and adenoviral (AV) vectors and tested for their ability to inhibit HCV core expression in a tissue culture model. RESULTS: Several Rzs targeting highly conserved HCV sequences effectively degraded positive and negative strands of HCV RNA in vitro. Furthermore, substantial inhibition of HCV gene expression was observed in tissue culture using viral vectors to deliver and express Rz genes. CONCLUSIONS: Rz gene therapy has potential for the production of anti-viral drugs directed against HCV. Initial studies employing Rz gene therapy to produced anti-viral drugs against HCV have proved successful. Rz gene therapy may be a useful approach to overcome problems associated with anti-HCV drug design, such as the emergence of drug-resistant mutants.

Adenoviridae↗
PubMed2001

Clinical protocol. A phase 1 open-label clinical trial of the safety and tolerability of single escalating doses of autologous CD4 T cells transduced with VRX496 in HIV-positive subjects.

The proposed study is a Phase I trial to evaluate a HIV-based lentiviral vector carrying an antisense sequence targeted to HIV in the treatment of HIV infection. The primary objective of this Phase I study is to determine the safety and tolerability of treatment with autologous CD4+ T cells modified (transduced) ex vivo with VRX496 when administered to HIV-infected patients. VRX496 is a completely gutted lentiviral vector and does not code for any viral proteins. The viral vector contains an antisense sequence targeted to the HIV envelope (env) gene. VRX496 directly interferes with wild-type HIV (wt-HIV) expression via anti-env antisense expression in vector transduced CD4 cells that become infected with wt-HIV. Expression of the anti-HIV antisense env from a HIV vector transcript would target wt-HIV RNA and destroy it, and hence, decrease productive HIV replication from CD4 T cells. The clinical goal for this treatment approach is to decrease viral loads and promote CD4 T cell survival in vivo. Data from in vitro studies suggest that HIV vectors such as VRX496 could potentially reduce viral loads in HIV-infected individuals and thus could delay the onset to AIDS while promoting CD4 T cell survival and providing the immune system with a better chance to control the infection. Additionally, preliminary results from experiments in SCID mice (mice with transplanted human immune cells) indicate that the human cells transduced with VRX496 and implanted into the SCID mice do not elicit any overt adverse effects. HIV-infected patients (CD4 T cell count of >200/mm3, discontinued from HAART therapy) will undergo leukoapheresis with subsequent CD4 T cell isolation. Patient CD4 T cells will be transduced ex vivo with the vector, expanded for 8-11 days, and then the modified cells will be reintroduced into the patient. Each subject will receive a single intravenous injection infused over 30 minutes; subjects will be examined 24, 48, and 72 hours post-injection and weekly for 4 weeks. Patients will receive one of four different ascending doses (1 x 10(9), 3 x 10(9), 1 x 10(10), and 3 X 10(10) cells/patient). Doses will be administered to four independent, sequential subject cohorts of 3 patients. Groups will be administered escalating doses at 6-week intervals after safety has been demonstrated in the previous group. Follow-up examinations will be conducted 1, 3, and 6 months post-injection. Long term follow-up including RCR testing will be performed.

Adolescent↗
PubMed1997

Enhancing efficacy of recombinant anticancer vaccines with prime/boost regimens that use two different vectors.

BACKGROUND: The identification of tumor-associated antigens and the cloning of DNA sequences encoding them have enabled the development of anticancer vaccines. Such vaccines target tumors by stimulating an immune response against the antigens. One method of vaccination involves the delivery of antigen-encoding DNA sequences, and a number of recombinant vectors have been used for this purpose. To optimize the efficacy of recombinant vaccines, we compared primary and booster treatment regimens that used a single vector (i.e., homologous boosting) with regimens that used two different vectors (i.e., heterologous boosting). METHODS: Pulmonary tumors (experimental metastases) were induced in BALB/c mice inoculated with CT26.CL25 murine colon carcinoma cells, which express recombinant bacterial beta-galactosidase (the model antigen). Protocols for subsequent vaccination used three vectors that encoded beta-galactosidase--vaccinia (cowpox) virus, fowlpox virus, naked bacterial plasmid DNA. Mouse survival was evaluated in conjunction with antibody and cytotoxic T-lymphocyte responses to beta-galactosidase. RESULTS: Heterologous boosting resulted in significantly longer mouse survival than homologous boosting (all P<.0001, two-sided). Potent antigen-specific cytotoxic T lymphocytes were generated following heterologous boosting with poxvirus vectors. This response was not observed with any of the homologous boosting regimens. Mice primed with recombinant poxvirus vectors generated highly specific antibodies against viral proteins. CONCLUSIONS: The poor efficacy of homologous boosting regimens with viral vectors was probably a consequence of the induction of a strong antiviral antibody response. Heterologous boosting augmented antitumor immunity by generating a strong antigen-specific cytotoxic T-lymphocyte response. These data suggest that heterologous boosting strategies may be useful in increasing the efficacy of recombinant DNA anticancer vaccines that have now entered clinical trials.

Animals↗
PubMed2006

Cellular and genetic therapies for haemophilia.

Haemophilia continues to be a prime target for a variety of gene and cell-based therapies. Pre-clinical successes in both mouse and dog models of the disease have been documented with a variety of approaches over the past decade, and there have now been six small clinical trials of gene transfer in haemophilia. To date, the only significant adverse events documented in these trials have been related to host immune responses, indicating that immunologic barriers continue to represent the major obstacle to achieving success of gene transfer in humans. Despite these challenges, new strategies are being explored with novel serotypes of viral vectors and with the use of transient periods of immunosuppression to attenuate the immune response to the vector and transgene product following gene delivery. Two new clinical trials, both using AAV vectors, will likely start within the next year, and additional large animal pre-clinical studies using other viral vector-mediated approaches for gene transfer are expected in the near future.

Animals↗
PubMed2003

Simultaneous on/off regulation of transgenes located on a mammalian chromosome with Cre-expressing adenovirus and a mutant loxP.

The site-specific recombinase Cre has often been used for on/off regulation of expression of transgenes introduced into the mammalian chromosome. However, this method is only applicable to the regulation of a single gene and cannot be used to simultaneously regulate two genes, because site-specific recombination occurs from the target loxP sequence of one regulating unit to the loxP sequence of any other unit and would eventually disrupt the structure of both regulating units. We previously reported a mutant loxP sequence with a two base substitution called loxP V (previously called loxP 2272), with which wild-type loxP cannot recombine but with which the identical mutant loxP recombines efficiently. In the present study we isolated cell lines bearing two regulating units on a chromosome containing a pair of wild-type loxP sequences or mutant loxP V sequences. After infection with Cre-expressing recombinant adenovirus AxCANCre, expression of a gene in each regulating unit was simultaneously turned on and off. Southern analyses showed that both regulating units were processed simultaneously and independently, even after infection with a limited amount of AxCANCre. The results showed that simultaneous regulation of gene expression on a mammalian chromosome mediated by Cre can be achieved by using mutant loxP V and wild-type loxP. This method may be a useful approach for conditional transgenic/knockout animals and investigation of gene function involving two genes simultaneously. Another possible application is for preparation of a new packaging cell line of viral vectors through simultaneous production of toxic viral genes.

Adenoviridae↗
PubMed1996

Inducible gene expression from defective herpes simplex virus vectors using the tetracycline-responsive promoter system.

Herpes simplex virus-based amplicon vectors have been used for gene transfer into cultured neurons and the adult CNS. Since constitutive expression of a foreign gene or overexpression of an endogenous gene may have deleterious effects, the ability to control temporal expression would be advantageous. To achieve inducible gene expression, we have incorporated the tetracycline-responsive promoter system into amplicon vectors and showed, both in vitro and in vivo, that expression can be modulated by tetracycline. Using the firefly luciferase as the reporter gene, maximal repression by tetracycline in hippocampal cultures was about 50-fold. Withdrawal of tetracycline derepressed gene expression, reaching maximal levels within 10-12 h. In contrast, addition of tetracycline to cultures without prior tetracycline exposure inhibited gene expression rapidly; luciferase activity was reduced to less than 8% within 24 h. In adult rat hippocampus, vectors expressing luciferase or the Escherichia coli lacZ were repressed by tetracycline 9- and 60-fold, respectively. Maximum gene expression from the vectors occurred 2-3 days post-infection and declined thereafter. Such decline impeded further induction of expression by withdrawing tetracycline. This study demonstrates the feasibility of incorporating a powerful inducible promoter system into HSV vectors. The development of such an inducible viral vector system for gene transfer into the adult CNS might prove to be of experimental and therapeutic value.

Animals↗
PubMed2001

Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging.

Viral vectors based on lentiviruses, such as the human immunodeficiency virus, are able to transduce a broad spectrum of nondividing cells in vivo. This ability of lentiviral vectors makes them an attractive vehicle for gene transfer into the liver. In order to determine the requirements for efficient lentiviral gene transfer, we used a fluorescence imaging system, which allows the detection of cells and tissues that express fluorescent reporter genes (e.g., green fluorescence protein) in the living animal. We show that the latest generation of lentiviral vectors efficiently transduces the murine liver. Further analysis demonstrated that neither cell-cycle activation nor division of liver cells is a prerequisite for lentiviral gene transfer in vivo.

Animals↗
PubMed2005

Immune responses to adeno-associated virus vectors.

One of the biggest challenges in optimizing viral vectors for gene therapy relates to the immune response of the host. Adeno-associated virus (AAV) vectors are associated with low immunogenicity and toxicity, resulting in vector persistence and long-term transgene expression. The inability of AAV vectors to efficiently transduce or activate antigen presenting cells (APCs) may account for their decreased immunogenicity. AAV mediated gene therapy however, leads to the development of antibodies against the vector capsid. Anti-AAV antibodies have neutralizing effects that decrease the efficiency of in vivo gene therapy and can prevent vector re-administration. Furthermore, recent studies have shown that AAV vectors can elicit both cellular and humoral immune responses against the transgene product. Both cell-mediated response and humoral response to the delivered gene depend on a number of variables; including the nature of the transgene, the promoter used, the route and site of administration, vector dose and host factors. The response of the host to the vector, in terms of antigen-specific immunity, will play a substantial role in clinical outcome. It is therefore important to understand both, why AAV vectors are able to escape immunity and the circumstances and mechanisms that lead to the induction of immune responses. This review will summarize innate and adaptive immune responses to AAV vectors, discuss possible mechanisms and outline strategies, such as capsid modifications, use of alternative serotypes, or immunosuppression, which have been used to circumvent them.

Animals↗
PubMed1993

Increased viral titer through concentration of viral harvests from retroviral packaging lines.

Dependent on the viral vector and the specific assay used, viral titers produced from commonly used retroviral packaging cell lines have an upper limit in the range of 10(5) to 10(7) infectious units/ml. We have developed a generally applicable method, using hollow-fiber filtration technology, which allows for the concentration of infectious virus derived from packaging lines. This method resulted in a reproducible 10- to 30-fold increase in viral titer and can readily be scaled to accommodate larger input volumes. Over 80% of the input virus is recovered in an infectious form in the concentrate. Concentrated virus containing media was seen to produce higher infection frequencies in Jurkat T cells as compared to unconcentrated virus containing media; however, this was not proportional to the differences in viral titer observed by limiting dilution analysis on NIH-3T3 cells. These results are discussed in relation to the importance of factors other than viral titer in determining transduction frequencies.

3T3 Cells↗
PubMed1995

Delivery of herpesvirus and adenovirus to nude rat intracerebral tumors after osmotic blood-brain barrier disruption.

The delivery of viral vectors to the brain for treatment of intracerebral tumors is most commonly accomplished by stereotaxic inoculation directly into the tumor. However, the small volume of distribution by inoculation may limit the efficacy of viral therapy of large or disseminated tumors. We have investigated mechanisms to increase vector delivery to intracerebral xenografts of human LX-1 small-cell lung carcinoma tumors in the nude rat. The distribution of Escherichia coli lacZ transgene expression from primary viral infection was assessed after delivery of recombinant virus by intratumor inoculation or intracarotid infusion with or without osmotic disruption of the blood-brain barrier (BBB). These studies used replication-compromised herpes simplex virus type 1 (HSV; vector RH105) and replication-defective adenovirus (AdRSVlacZ), which represent two of the most commonly proposed viral vectors for tumor therapy. Transvascular delivery of both viruses to intracerebral tumor was demonstrated when administered intraarterially (i.a.) after osmotic BBB disruption (n = 9 for adenovirus; n = 7 for HSV), while no virus infection was apparent after i.a. administration without BBB modification (n = 8 for adenovirus; n = 4 for HSV). The thymidine kinase-negative HSV vector infected clumps of tumor cells as a result of its ability to replicate selectively in dividing cells. Osmotic BBB disruption in combination with i.a. administration of viral vectors may offer a method of global delivery to treat disseminated brain tumors.

Adenoviridae↗
PubMed2004

Recent advances in liver-directed gene transfer vectors.

BACKGROUND: Gene therapy as part of modern molecular medicine holds great promise for the treatment of hepatocellular carcinoma (HCC) and has the potential to bring a revolutionary era to cancer treatment. For the past decade various viral and non-viral vectors have been engineered for improved liver gene therapy. DATA RESOURCES: An English-language literature search using MEDLINE (2004), Index Medicus (2004) and bibliographic reviews of books and review articles. Liver-directed gene transfer vectors and their history and recent clinical applications. RESULTS: The ultimate goal of liver-directed gene therapy for HCC is the stable expression of a therapeutic transgene in a significant proportion of hepatocytes. The design of a vector system providing efficient and stable gene engraftment and expression in human hepatocytes is still a challenging issue. The advantages and disadvantages of the genetically engineered vector of viral or non-viral origin are discussed with respect to their essential relevance. CONCLUSION: Liver gene therapy has a long way to go and efficient and innocuous liver-directed gene transfer vectors are therefore urgently required.

Animals↗
PubMed2001

Calbindin d28k overexpression protects striatal neurons from transient focal cerebral ischemia.

BACKGROUND AND PURPOSE: Increased intracellular calcium accumulation is known to potentiate ischemic injury. Whether endogenous calcium-binding proteins can attenuate this injury has not been clearly established, and existing data are conflicting. Calbindin D28K (CaBP) is one such intracellular calcium buffer. We investigated whether CaBP overexpression is neuroprotective against transient focal cerebral ischemia. METHODS: Bipromoter, replication-incompetent herpes simplex virus vectors that encoded the genes for cabp and, as a reporter gene, lacZ were used. Sprague-Dawley rats received bilateral striatal injections of viral vector 12 to 15 hours before ischemia onset. With the use of an intraluminal occluding suture, animals were subjected to 1 hour of middle cerebral artery occlusion followed by 47 hours of reperfusion. Brains were harvested and stained with X-gal (to visualize beta-galactosidase, the gene product of lacZ). The number of remaining virally transfected, X-gal-stained neurons in both the ischemic and contralateral striata were counted and expressed as the percentage of surviving neurons in the ischemic striatum relative to the contralateral nonischemic striatum. RESULTS: Striatal neuron survivorship among cabp-injected animals was 53.5+/-4.1% (n=10) versus 26.8+/-5.4% among those receiving lacZ (n=9) (mean+/-SEM; P<0.001). CONCLUSIONS: We conclude that viral vector-mediated overexpression of CaBP leads to neuroprotection in this model of central nervous system injury. This is the first demonstration that CaBP overexpression protects neurons in a focal stroke model.

Animals↗