Potential Vector For Gene Therapy Research Articles

Foamy Virus Integrase in Development of Viral Vector for Gene Therapy.

Due to the broad host suitability of viral vectors and their high gene delivery capacity, many researchers are focusing on viral vector-mediated gene therapy. Among the retroviruses, foamy viruses have been considered potential gene therapy vectors because of their non-pathogenicity. To date, the prototype foamy virus is the only retrovirus that has a high-resolution structure of intasomes, nucleoprotein complexes formed by integrase, and viral DNA. The integration of viral DNA into the host chromosome is an essential step for viral vector development. This process is mediated by virally encoded integrase, which catalyzes unique chemical reactions. Additionally, recent studies on foamy virus integrase elucidated the catalytic functions of its three distinct domains and their effect on viral pathogenicity. This review focuses on recent advancements in biochemical, structural, and functional studies of foamy virus integrase for gene therapy vector research.

Astrocyte-selective AAV gene therapy through the endogenous GFAP promoter results in robust transduction in the rat spinal cord following injury

Adeno-associated viral (AAV) vectors are a promising system for transgene delivery into the central nervous system (CNS) based on their safety profile and long-term gene expression. Gene delivery to the CNS has largely been neuron centric but advances in AAV technology are facilitating the development of approaches to enable transduction of glial cells. Considering the role of astrocytes in the on-going secondary damage in spinal cord injury (SCI), an AAV vector that targets astrocytes could show benefit as a potential treatment. Transduction efficiency, transgene expression and cellular tropism were compared for the AAV serotypes AAV5, AAV9 and AAVRec2 whereby destabilised yellow fluorescent protein (dYFP) was controlled by the GFAP or the truncated GfaABC1D promoter. The vectors were tested in primary spinal cord astrocyte cell culture, spinal cord slice culture and an in vivo model of SCI contusion. AAV5 resulted in greater transduction efficiency, transgene expression and astrocyte tropism compared with AAV9 and AAVRec2. In a rodent model of SCI, robust transgene expression by AAV5-GFAP/GfaABC1D-dYFP was observed through 12 mm of spinal cord tissue and expression was largely restricted to astrocytes. Thus, AAV5-GFAP/GfaABC1D carries the potential as a potential gene therapy vector, particularly for transducing astrocytes in the damaged spinal cord.

Amsacta moorei entomopoxvirus encodes a functional heparin-binding glycosyltransferase (AMV248).

Amsacta moorei entomopoxvirus (AMEV) infects certain lepidopteran and orthopteran insects and is the most studied member of the genus Betaentomopoxvirus. It has been considered as a potential vector for gene therapy, a vector to express exogenous proteins and a biological control agent. One of its open reading frames, amv248, encodes a putative glycosyltransferase and is the only known attachment protein conserved in AMEV and chordopoxviruses. The ORF was successfully expressed and the protein was shown to bind soluble heparin, both in silico and in vitro. Our results also showed that, while viral infection was inhibited by soluble glycosaminoglycans (GAGs), GAG-deficient cells were more resistant to the virus. Finally, we revealed that amv248 encodes an active heparin-binding glycosyltransferase which is likely to have a key role in the initiation of infection by AMEV.

Neutralizing antibodies against AAV2, AAV5and AAV8 in healthy and HIV-1-infected subjects in China: implications for gene therapy using AAV vectors.

Adeno-associated viruses (AAV) have attracted attention as potential vectors for gene therapy and vaccines against several diseases, including HIV-1 infection. However, the presence of neutralizing antibodies (NAbs) after natural AAV infections inhibits their transfection in re-exposed subjects. To identify candidate AAV vectors for therapeutic or prophylactic HIV vaccines, NAbs against AAV2, AAV5 and AAV8 were screened in the sera of healthy individuals in China and 10 developed countries and an HIV-1-infected Chinese population. Seroprevalence was higher for AAV2 (96.6%) and AAV8 (82.0%) than for AAV5 (40.2%) in normal Chinese subjects. Among individuals seropositive for AAV5, >80% had low NAb titers (<1:90). The prevalence and titers of NAbs against the three AAVs were significantly higher in China than in developed countries (P<0.01). The prevalence of NAbs against AAV5 did not differ significantly between healthy and HIV-1-infected Chinese subjects (P=0.39). Co-occurrence of NAbs against AAV2, AAV5, and AAV8 was observed in the healthy population, and 15, 41, and 41% of individuals were AAV2(+), AAV2(+)/AAV8(+), and AAV2(+)/AAV5(+)/AAV8(+), respectively. Therefore, AAV5 exposure is low in healthy and HIV-1-infected populations Chinese individuals, and vectors based on AAV5 may be appropriate for human gene therapy or vaccines.

Ex vivointracoronary gene transfer of adeno-associated virus 2 leads to superior transduction over serotypes 8 and 9 in rat heart transplants

Heart transplant gene therapy requires vectors with long-lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno-associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar-Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR-, RT-PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies.

RNA viruses and microRNAs: challenging discoveries for the 21st century

RNA viruses represent the predominant cause of many clinically relevant viral diseases in humans. Among several evolutionary advantages acquired by RNA viruses, the ability to usurp host cellular machinery and evade antiviral immune responses is imperative. During the past decade, RNA interference mechanisms, especially microRNA (miRNA)-mediated regulation of cellular protein expression, have revolutionized our understanding of host-viral interactions. Although it is well established that several DNA viruses express miRNAs that play crucial roles in their pathogenesis, expression of miRNAs by RNA viruses remains controversial. However, modulation of the miRNA machinery by RNA viruses may confer multiple benefits for enhanced viral replication and survival in host cells. In this review, we discuss the current literature on RNA viruses that may encode miRNAs and the varied advantages of engineering RNA viruses to express miRNAs as potential vectors for gene therapy. In addition, we review how different families of RNA viruses can alter miRNA machinery for productive replication, evasion of antiviral immune responses, and prolonged survival. We underscore the need to further explore the complex interactions of RNA viruses with host miRNAs to augment our understanding of host-virus interplay.

Transfection using hydroxyapatite nanoparticles in the inner ear via an intact round window membrane in chinchilla

Hydroxyapatite nanoparticles (nHAT) are known to have excellent biocompatibility, and have attracted increasing attention as new candidates of non-viral vectors for gene therapy. In our previous studies, nHAT carrying a therapeutic gene and a reporter gene were successfully transfected into the spiral ganglion neurons in the inner ear of guinea pigs in vivo as well as in the cultured cell lines, although the transfection efficiencies were never higher than 30%. In this study, the surface modification of nHAT with polyethylenimine (PEI) was made (PEI–nHAT, diameter = 73.09 ± 27.32 nm) and a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) gene and neurotrophin-3 (NT-3) gene was constructed as pEGFPC2–NT3. The PEI modified nHAT and the recombinant plasmid was then connected to form the nHAT-based vector–gene complex (PEI–nHAT–pEGFPC2–NT3). This complex was then placed onto the intact round window membranes of the chinchillas for inner ear transfection. Auditory brainstem response (ABR) was tested to evaluate auditory function. Green fluorescence of EGFP was observed using confocal microscopy 48 h after administering vector–gene complexes. There was no significant threshold shift in tone burst-evoked ABR at any tested frequency. Abundant, condensed green fluorescence was found in dark cells on both sides of the crista and around the macula of the utricle. Scattered EGFP signals were also detected in vestibular hair cells, some Schwann cells in the cochlear spiral ganglion region, some outer pillar cells in the organ of Corti, and a few cells in the stria vascularis. The density of green fluorescence-marked cells was obviously higher in the vestibular dark cell area than in other areas of the inner ear, suggesting that vestibular dark cells may have the ability to actively engulf the nHAT-based vector–gene complexes. Considering the high transfection efficiency in the vestibular system, PEI–nHAT may be a potential vector for gene therapy of inner ear diseases, especially vestibular disorders, and deserves further study.

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High Cooperativity of the SV40 Major Capsid Protein VP1 in Virus Assembly

SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of ∼6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility.

PEO−PPO Block Copolymer Vectors Do Not Interact Directly with DNA but with Lipid Membranes

Small angle neutron (SANS) and light scattering was used to study the interaction between fragments of double stranded deoxyribonucleic acid (DNA) and a synthetic triblock [poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)] amphiphilic polymer, known as L64, a potential vector for gene therapy. The mechanism of action of this vector is yet unknown. The contrast variation method was used to separate the partial structure factors of the different components in mixtures of triblock and DNA. It has been found that the copolymer and DNA molecules exhibit repulsive interactions. Further, the interaction between the copolymer and a model lipid membrane was investigated in order to explain the action of the vector. Electrical measurements on black lipid membranes indicated that the main effect of L64 as a vector is to permeabilize the cell's membrane.

Integrating DNA Vectors For Gene Therapy

The many platform and poster presentations at the last annual meeting of the American Society for Gene Therapy (ASGT) make it clear that nonviral, integrating DNAs have reached a tipping point in establishing themselves as potential vectors for gene therapy.1 These vectors currently include the Sleeping Beauty (SB) transposon system,2 the first such vector used in gene therapy experiments,3 the ϕC31 and ϕBT1 integrase systems from Streptomyces,4, 5 and more recently the insect piggyBac6 and fish Tol27 transposon systems.

Calcium Phosphate Nanoparticles as a Novel Nonviral Vector for Efficient Transfection of DNA in Cancer Gene Therapy

To explore an efficient gene vector in cancer gene therapy, a novel nonviral vector calcium phosphate nanoparticle (CPNP) was developed. Transmission electromicroscopy and Zeta potential demonstrated that CPNP was 23.5-34.5 nm diameters and had +16.8 mV positive surface charges. The analysis of the CPNPDNA complex showed CPNP could transfer foreign DNA into targeted cells with high transfection efficiency, as well as its DNA-binding property and protection of DNA from degradation. Furthermore, the CPNP-DNA complex had no obvious cytotoxicity for CNE-2 cells, while the liposome-DNA complex had certain cytotoxicity. When the CPNP combined with suicide genes yCDglyTK for nasopharyngeal carcinoma (NPC) therapy in vitro, just 24.76% of cells survived when the wild-type CNE-2 cells were treated with the CPNP-yCDglyTK complex plus the prodrug, 5-FC (200 mg/mL). Otherwise, the expression of yCDglyTK was detected in implanted CNE-2 tumors by reverse transcription-polymerase chain reaction (RT-PCR) analysis when the CNE-2 tumor was treated with an intratumoral injection of the CPNPyCDglyTK complex. Our results showed that the CPNP might be a potential vector for gene therapy.

DNA-Induced Aggregation of Zwitterionic Oligolamellar Liposome

Liposome consisting of a single zwitterionic lipid as the potential vector for gene therapy has been reported recently; however, whether polyanionic DNA can bind directly with zwitterionic lipid without the aid of multivalent salt still remains unresolved. In this study, we reveal the aggregation of zwitterionic oligolamellar liposomes composed of 1,2-di(cis-9-octadecenoyl)-sn-glycero-3-phosphocholine induced by DNA without the presence of multivalent salt. Our results demonstrate that only a small fraction (<10%) of DNA can bind electrostatically with a portion of the liposomes. Such a low degree of binding, however, induces significant aggregation of these oligolamellar liposomes, yielding large multilamellar particles in which the number of hydrophilic/hydrophobic layer stacking becomes sufficiently large to yield multiple diffraction peaks in the small-angle X-ray scattering profile. Addition of monovalent salt such as NaCl tends to disrupt the multilamellar structure.

Suicide gene therapy for human oral squamous cell carcinoma cell lines with adeno-associated virus vector.

The purpose of this study was to test the possibility of gene transfer as a new therapy for oral cancer. Adeno-associated virus (AAV) has already been used in the fields of cystic fibrosis and Parkinson's disease as a potential vector for gene therapy because of its wide host range, high transduction efficiency, and lack of cytopathogenicity. Four human oral squamous cell carcinoma cell lines were transduced with an AAV vector containing the beta-galactosidase gene (AAVlacZ) in vitro. Gene transduction efficiency was from 20 to 50% at a multiplicity of infection (MOI; for the purposes of this study the number of vector genomes per target cell) of 1x10(3), and nearly 100% of each cell line were transduced at an MOI of 1x10(4). Next, four cell lines were transduced with an AAV vector containing the herpes simplex virus thymidine kinase (HSVtk) gene, which sensitizes transduced cells to ganciclovir (GCV). Subsequent administration of GCV resulted in nearly 100% tumor cell killing at an MOI of 1x10(4) and from 70 to 80% tumor cell killing at an MOI of 1x10(3). These results suggest that AAV-mediated gene transfer of HSVtk and administration of GCV has potential as a new therapy for oral squamous cell carcinoma.

Muscle-specific promoters may be necessary for adeno-associated virus-mediated gene transfer in the treatment of muscular dystrophies.

Recombinant adeno-associated virus (rAAV) vectors allow efficient gene transfer and expression in the muscle; therefore, rAAVs represent a potential gene therapy vector for muscular dystrophies. For further investigations, we used a mouse muscular dystrophy model (gsg(-/-) mice) gamma-sarcoglycan, a subunit of the dystrophin-glycoprotein complex, is missing. gsg(-/-) mice develop progressive dystrophy representative of a severe human phenotype disease. We previously showed high levels and stable expression of gamma-sarcoglycan in myofibers after direct muscle injection into gsg(-/-) mice of a recombinant AAV vector (AAV.dMCK.gSG) carrying the gamma-sarcoglycan cDNA driven by a muscle-specific promoter (truncated version of muscle creatine kinase). Here, we show that when gamma-sarcoglycan expression is driven by the ubiquitous cytomegalovirus (CMV) promoter (AAV.CMV.gSG), lower levels of transgene expression are observed and are associated with a humoral response to gamma-sarcoglycan. When using an rAAV vector, expressing the highly immunogenic product gamma-galactosidase under the CMV promoter (AAV.CMV.LacZ), we measured a strong cellular and humoral immune response to the transgene after intramuscular injection into gsg(-/-) mice. This study suggests that restriction of transgene expression to the muscle is an important criterion for the treatment of muscular dystrophies and will aid in the design of protocols for gene therapy.

Rescue of skeletal muscles of gamma-sarcoglycan-deficient mice with adeno-associated virus-mediated gene transfer.

In humans, a subset of cases of Limb-girdle muscular dystrophy (LGMD) arise from mutations in the genes encoding one of the sarcoglycan (alpha, beta, gamma, or delta) subunits of the dystrophin-glycoprotein complex. While adeno-associated virus (AAV) is a potential gene therapy vector for these dystrophies, it is unclear if AAV can be used if a diseased muscle is undergoing rapid degeneration and necrosis. The skeletal muscles of mice lacking gamma-sarcoglycan (gsg-/- mice) differ from the animal models that have been evaluated to date in that the severity of the skeletal muscle pathology is much greater and more representative of that of humans with muscular dystrophy. Following direct muscle injection of a recombinant AAV [in which human gamma-sarcoglycan expression is driven by a truncated muscle creatine kinase (MCK) promoter/enhancer], we observed significant numbers of muscle fibers expressing gamma-sarcoglycan and an overall improvement of the histologic pattern of dystrophy. However, these results could be achieved only if injections into the muscle were prior to the development of significant fibrosis in the muscle. The results presented in this report show promise for AAV gene therapy for LGMD, but underscore the need for intervention early in the time course of the disease process.

Properties of human foamy virus relevant to its development as a vector for gene therapy.

The Spumaviridae (foamy viruses) are increasingly being considered as potential vectors for gene therapy, yet little has been documented of their basic cell biology. This study demonstrates that human foamy virus (HFV) has a broad tropism and that the receptor for HFV is expressed not only on many mammalian, but on avian and reptilian cells. Receptor interference assays using an envelope-expressing cell line and a vesicular stomatitis virus/HFV pseudotype virus demonstrate that the cellular receptor is common to all primate members of the genus. The majority of foamy virus particles assemble and remain sequestered intracellularly. A rapid and quantitative method of assaying foamy virus infectivity by reverse transcriptase activity facilitates the use of classical protocols to increase infectious virus titres in vitro to > or = 10(6) TCID/ml.

Mycoplasma: a new potential vector for gene therapy?

The introduction of foreign genetic material in living cells is the basis of the current protocols of gene therapy. The concept that the de novo synthesis of a foreign therapeutic protein requires the entrance of the corresponding gene into target cells via virus or synthetic vectors is directly inherited from experiments on bacterial transduction or transformation. However, the difficulties inherent in the penetration and the expression of foreign DNA into eucaryotic cells are probably responsible for the low efficiency of this therapeutic approach. In this paper, we explore the possibility of avoiding these limiting critical steps by expressing the foreign gene on the surface rather than inside the target cells by the use of mycoplasma, the smallest reported living cell. The absence of transfer of genetic information between this vector and eucaryotic target cells, the sensibility of mycoplasmas to antibiotics and their cytadherance are among the interesting features of this potential vector. The interest of this new vector in the case, e.g. of the gene-directed enzyme prodrug therapy of solid tumors, is discussed.

Generation and Use of Recombinant Reporter Viruses for Study of Herpes Simplex Virus Infectionsin Vivo

It has become increasingly clear that the fate of herpes simplex virus (HSV) infections involves complex interactions between the virus and the specific cell types that comprise the tissues of the animal host. No reliable cell culture system for studying the establishment of latency and reactivation exists, and therefore these studies must be performed within animal models. One difficulty in elucidating the molecular regulation of these events is in determining the transcriptional activity of key viral genes during different stages of the infectionin vivo.The heterogeneous cell types comprising infected tissues make PCR analysis of tissue homogenates difficult to interpret. The need to characterize expression of multiple transcriptional markers reliably and quantitatively at the level of individual cells is therefore key to determining the interplay between viral and cellular genes during latency and reactivation. Here we discuss the construction and evaluation of HSV reporter viruses that have been used in these analyses. HSV-1 recombinants have been engineered with representative viral promoters driving β-galactosidase as a reporter. Methodology used to evaluate the levels of gene expression using (1) quantitative enzyme assays, (2) precipitatable substrate assays to localize the positive cells, and (3) immunohistochemistry and fluorescence assays to look at colocalization of markers duringin vivoinfection is presented. In addition to studying the molecular pathogenesis of HSV, the application of similar reporter viruses to evaluate promoters for targeting various differentiated tissues will be useful in developing these viruses as potential vectors for gene therapy.

Marrow stromal cells as stem cells for continual renewal of nonhematopoietic tissues and as potential vectors for gene therapy.

Marrow stromal cells as stem cells for continual renewal of nonhematopoietic tissues and as potential vectors for gene therapy.