Ability Of Vectors Research Articles

A Simple and Versatile Method for Ex Vivo Monitoring of Goat Vaginal Mucosa Transduction by Viral Vector Vaccines.

Goat may represent a valid large animal model for human pathogens and new vaccines testing. Appropriate vaccine administration is a critical component of a successful immunization program. The wrong route of administration may reduce the efficacy of the vaccine, whereas the proper administration strategy can enhance it. Viral vectors have been employed successfully for goat and sheep immunization; however, no data concerning the vaginal route are available. A viral vector's ability to transduce the site of inoculation is of primary interest. In this study, a fast and reliable ex vivo assay for testing the transduction capability of an Ad5-based vector when intravaginally administered was developed. An Ad5 vector delivering an expression cassette with a bicistronic reporter gene, Ad5-CMV-turboGFP-IRES-Luc2, was constructed. We demonstrated Ad5-CMV-turboGFP-IRES-Luc2's ability to transduce caprine vaginal mucosa by ex vivo bioluminescent imaging (BLI) employing a simple CCD camera apparatus for chemiluminescence western immunoblotting. These data, though simple, provide valuable insights into developing a vaginal immunization strategy using a viral vector-based vaccine to protect against pathogens causing genital diseases.

Host blood meal identity modifies vector gene expression and competency.

A vector's susceptibility and ability to transmit a pathogen—termed vector competency—determines disease outcomes, yet the ecological factors influencing tick vector competency remain largely unknown. Ixodes pacificus, the tick vector of Borrelia burgdorferi (Bb) in the western U.S., feeds on rodents, birds, and lizards. Rodents and birds are reservoirs for Bb and infect juvenile ticks, while lizards are refractory to Bb and cannot infect feeding ticks. Additionally, the lizard bloodmeal contains borreliacidal properties, clearing previously infected feeding ticks of their Bb infection. Despite I. pacificus feeding on a range of hosts, it is undetermined how the host identity of the larval bloodmeal affects future nymphal vector competency. We experimentally evaluate the influence of larval host bloodmeal on Bb acquisition by nymphal I. pacificus. Larval I. pacificus were fed on either lizards or mice and after molting, nymphs were fed on Bb‐infected mice. We found that lizard‐fed larvae were significantly more likely to become infected with Bb during their next bloodmeal than mouse‐fed larvae. We also conducted the first RNA‐seq analysis on whole‐bodied I. pacificus and found significant upregulation of tick antioxidants and antimicrobial peptides in the lizard‐fed group. Our results indicate that the lizard bloodmeal significantly alters vector competency and gene regulation in ticks, highlighting the importance of host bloodmeal identity in vector‐borne disease transmission and upends prior notions about the role of lizards in Lyme disease community ecology.

An integrative approach to symbiont-mediated vector control for agricultural pathogens.

Vector-borne pathogens pose significant threats to agricultural productivity. Methods that exploit associations between insects and their symbiotic microbes, dubbed symbiont-mediated vector control, are emerging as viable alternatives to insecticides for the control of vector-borne agricultural plant pathogens. The development of methods for effective microbial manipulation, such as RNA interference and paratransgenesis, may facilitate symbiont-mediated vector control tactics aimed at either suppressing insect populations or at manipulating vector competence, an insect vector's ability to acquire, harbor, and transmit pathogens. As suppression strategies transition from the laboratory to the field, the need for methods to evaluate their viability and predict their outcomes is apparent. Mathematical models of symbiont impact on agricultural disease can inform the development of symbiont-mediated vector control. We propose an integrative approach, combining theoretical and empirical experiments to identify the best practices for achieving meaningful improvements to crop health and productivity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine.

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector's ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

129. Does Transcription Influence AAV-Mediated Homologous Recombination?

Recombinant adeno-associated viral (AAV) vectors constitute one of the most promising tools for gene transfer. While the majority of AAV transduction events are episomal, our laboratory recently exploited the vector's ability to induce homologous recombination (HR) to design promoterless vectors that utilize chromosomal homology arms flanking a ribosomal skipping P2A-therapeutic coding sequence construct to integrate sequences just upstream of the stop codon of an endogenous gene. When targeting the albumin gene in the liver, a chimeric mRNA transcript capable of producing both albumin and a second therapeutic protein is consequently created. Not only do these vectors offer the permanence of gene transfer associated with integration, but a vector lacking a promoter reduces the chance for oncogene activation from off-target vector integration. AAV-mediated HR appears to be more efficient when targeting transcriptionally active loci, yet it is unclear if transcription itself or other factors that secondarily influence transcription, such as chromatin state, are directly linked to AAV-mediated HR. We therefore set out to establish if the transcriptional rate in particular is a major determinant for this type of HR.To do this, we developed a strategy to quantify precision AAV-mediated transgene integration by exploiting an engineered locus whose transcriptional rates could be controlled by drug administration. To this end, we used lentiviral vectors to generate a series of clonal HeLa cell populations each harboring a single-copy, doxycycline-inducible genomic site expressing the human alpha 1-antitrypsin (hAAT) cDNA. Upon induction with doxycycline, transgene expression from this site increases by greater than 1,000-fold. Each clonal cell population was infected with an AAV serotype DJ vector containing homology arms to the integrated hAAT cDNA and a P2A- codon-optimized human coagulation factor IX (hFIX) cDNA or P2A-eGFP construct that would allow for multicistronic genomic expression of both peptides from a single mRNA only if HR occurred. Transgene hFIX or eGFP expression as a measure of HR is established by quantitative RT-PCR and by transgene protein measurements. By modulating the rate of transcription just prior to rAAV vector administration, we can establish if transcription from a given genomic locus influences the frequency of AAV-mediated HR. Our preliminary results suggest that transcription per se might not be the predominant factor influencing the rate of HR. Further studies will provide more insight into the mechanism of gene targeting by AAV, optimal target site selection, and potentially expand the use of AAV-mediated gene targeting for treating various genetic and acquired diseases.

Comparative analysis of midgut bacterial communities in three aedine mosquito species from dengue-endemic and non-endemic areas of Rajasthan, India.

Dengue viruses are transmitted to humans through the bites of infected female aedine mosquitoes. Differences in the composition and structure of bacterial communities in the midguts of mosquitoes may affect the vector's ability to transmit the disease. To investigate and analyse the role of midgut bacterial communities in viral transmission, midgut bacteria from three species, namely Stegomyia aegypti (= Aedes aegypti), Fredwardsius vittatus (= Aedes vittatus) and Stegomyia albopicta (= Aedes albopictus) (all: Diptera: Culicidae), from dengue-endemic and non-endemic areas of Rajasthan, India were compared. Construction and analyses of six 16S rRNA gene libraries indicated that Serratia spp.-related phylotypes dominated all clone libraries of the three mosquito species from areas in which dengue is not endemic. In dengue-endemic areas, phylotypes related to Aeromonas, Enhydrobacter spp. and uncultivated bacterium dominated the clone libraries of S. aegypti, F. vittatus and S. albopicta, respectively. Diversity indices analysis and real-time TaqMan polymerase chain reaction assays showed bacterial diversity and abundance in the midguts of S. aegypti to be higher than in the other two species. Significant differences observed among midgut bacterial communities of the three mosquito species from areas in which dengue is and is not endemic, respectively, may be related to the vectorial capacity of mosquitoes to carry dengue viruses and, hence, to the prevalence of disease in some areas.

454. Prior AAV2-hIL10 Infusion Decreases AAV9-Dependent Immunotoxicity in Rat Brain

Cerebral infusion of AAV9 vectors encoding non-self proteins results in the initiation of a robust immune reaction at the site of injection due to the vector's ability to transduce antigen-presenting cells in the brain [1]. Infusion of AAV9 encoding a human gene, aromatic L-amino acid decarboxylase (hAADC), elicited an initial innate immune activation at the site of infusion that was succeeded by a robust adaptive immune response that included generation of hAADC antibodies, lymphocytic infiltration and cell-mediated elimination of transduced cells in the brain. In this study, we investigated whether the potent anti-inflammatory cytokine, human interleukin-10 (hIL10) could suppress this immune response. Four weeks prior to unilateral infusion of AAV9-hAADC into rat striatum, we infused AAV2-hIL10 bilaterally into striatum (control rats received bilaterally injection of AAV2-GFP). Six weeks after AAV9-hAADC injection, control rats revealed robust amphetamine-induced ipsilateral rotational behavior (956±203 (S.D.) turns/1 hour). In while, in AAV2-hIL10-treated rats ipsilateral rotations were reduced by 66 % compared with control rats and this effect persisted for at least 8 weeks. This neuroprotective effect of hIL10 was evident histologically. The number of surviving neurons (NeuN) was significantly increased in AAV2-hIL10-treated animals compared to AAV2-GFP-treated controls, and microglial and astrocytic activation was diminished. Whereas, in control rats, a significant number of hAADC-positive astrocytes was observed (GFAP/hAADC double fluorescent staining), astrocytic transduction in AAV2-hIL10-treated rats was almost completely suppressed. This somewhat puzzling result may be due to an effect of IL10 on astrocytic expression of an AAV9 receptor required for transduction. However, our findings suggest that expression of IL-10 in the brain may be a useful adjunctive therapy in the treatment of neurological diseases in which the expression of a foreign antigen is unavoidable.

Vector migration and dispersal rates for sylvatic Trypanosoma cruzi transmission

The spread of vector-borne diseases are greatly increased by a vector's ability to migrate. Recent studies of sylvatic Trypanosoma cruzi transmission have motivated the study of vector migration across geographic regions. Due to the natural mechanisms in which vector-borne diseases are transmitted between vectors and hosts, vector dispersal among different host populations is a critical factor in the ability of the parasite to be spread across large regions. In this study we develop a general framework for deriving large-scale, discrete-space migration rates from small-scale, continuous-space dispersal data. We identify three defining characteristics of vector migration: distance, preferred direction of dispersal, and strength of preference for a particular direction. We consider several migration scenarios in which vectors may have no preference for dispersal in a particular direction or may disperse with a preferred direction, such as northeast. We examine what effect preferred direction has on the migration rate, as well as use the local to global framework to calculate numerical estimates for vector migration rates for the primary vectors in the southeast U.S. and northern Mexico, Triatoma sanguisuga and Triatoma gerstaeckeri, based on biological and experimental data. Results from this study can be applied to metapopulation models for species that migrate.

Impact of vector dispersal and host-plant fidelity on the dissemination of an emerging plant pathogen.

Dissemination of vector-transmitted pathogens depend on the survival and dispersal of the vector and the vector's ability to transmit the pathogen, while the host range of vector and pathogen determine the breath of transmission possibilities. In this study, we address how the interaction between dispersal and plant fidelities of a pathogen (stolbur phytoplasma tuf-a) and its vector (Hyalesthes obsoletus: Cixiidae) affect the emergence of the pathogen. Using genetic markers, we analysed the geographic origin and range expansion of both organisms in Western Europe and, specifically, whether the pathogen's dissemination in the northern range is caused by resident vectors widening their host-plant use from field bindweed to stinging nettle, and subsequent host specialisation. We found evidence for common origins of pathogen and vector south of the European Alps. Genetic patterns in vector populations show signals of secondary range expansion in Western Europe leading to dissemination of tuf-a pathogens, which might be newly acquired and of hybrid origin. Hence, the emergence of stolbur tuf-a in the northern range was explained by secondary immigration of vectors carrying stinging nettle-specialised tuf-a, not by widening the host-plant spectrum of resident vectors with pathogen transmission from field bindweed to stinging nettle nor by primary co-migration from the resident vector's historical area of origin. The introduction of tuf-a to stinging nettle in the northern range was therefore independent of vector's host-plant specialisation but the rapid pathogen dissemination depended on the vector's host shift, whereas the general dissemination elsewhere was linked to plant specialisation of the pathogen but not of the vector.

Genetic manipulation of endosymbionts to control vector and vector borne diseases

Vector borne diseases (VBD) are on the rise because of failure of the existing methods of control of vector and vector borne diseases and the climate change. A steep rise of VBDs are due to several factors like selection of insecticide resistant vector population, drug resistant parasite population and lack of effective vaccines against the VBDs. Environmental pollution, public health hazard and insecticide resistant vector population indicate that the insecticides are no longer a sustainable control method of vector and vector-borne diseases. Amongst the various alternative control strategies, symbiont based approach utilizing endosymbionts of arthropod vectors could be explored to control the vector and vector borne diseases. The endosymbiont population of arthropod vectors could be exploited in different ways viz., as a chemotherapeutic target, vaccine target for the control of vectors. Expression of molecules with antiparasitic activity by genetically transformed symbiotic bacteria of disease-transmitting arthropods may serve as a powerful approach to control certain arthropod-borne diseases. Genetic transformation of symbiotic bacteria of the arthropod vector to alter the vector's ability to transmit pathogen is an alternative means of blocking the transmission of VBDs. In Indian scenario, where dengue, chikungunya, malaria and filariosis are prevalent, paratransgenic based approach can be used effectively.

Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy

Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro, the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vector's ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.

Anopheles mortality is both age- and Plasmodium-density dependent: implications for malaria transmission

BackgroundDaily mortality is an important determinant of a vector's ability to transmit pathogens. Original simplifying assumptions in malaria transmission models presume vector mortality is independent of age, infection status and parasite load. Previous studies illustrate conflicting evidence as to the importance of Plasmodium-induced vector mortality, but very few studies to date have considered the effect of infection density on mosquito survival.MethodsA series of three experiments were conducted, each consisting of four cages of 400-1,000 Anopheles stephensi mosquitoes fed on blood infected with different Plasmodium berghei ookinete densities per microlitre of blood. Twice daily the numbers of dead mosquitoes in each group were recorded, and on alternate days a sample of live mosquitoes from each group were dissected to determine parasite density in both midgut and salivary glands.ResultsSurvival analyses indicate that mosquito mortality is both age- and infection intensity-dependent. Mosquitoes experienced an initially high, partly feeding-associated, mortality rate, which declined to a minimum before increasing with mosquito age and parasite intake. As a result, the life expectancy of a mosquito is shown to be dependent on both insect age and the density of Plasmodium infection.ConclusionThese results contribute to understanding in greater detail the processes that influence sporogony in the mosquito, indicate the impact that parasite density could have on malaria transmission dynamics, and have implications for the design, development, and evaluation of transmission-blocking strategies.

Naturally occurring singleton residues in AAV capsid impact vector performance and illustrate structural constraints

SummaryVectors based on the adeno-associated virus are attractive and versatile vehicles for in vivo gene transfer. The virus capsid is the primary interface with the cell that defines many pharmacological, immunological and molecular properties. Determinants of these interactions are often restricted to a limited number of capsid amino acids. In this study, a portfolio of novel AAV vectors was developed following a structure-function analysis of naturally occurring AAV capsid isolates. Singletons, which are particular residues on the AAV capsid that were variable in otherwise conserved amino acid positions were found to impact on vector's ability to be manufactured or to transduce. Data for those residues that mapped to monomer-monomer interface regions on the particle structure suggested a role in particle assembly. The change of singleton residues to the conserved amino acid resulted in the rescue of many isolates that were defective upon initial isolation. This led to the development of an AAV vector portfolio that encompasses 6 different clades and 3 other distinct AAV niches. Evaluation of the in vivo gene transfer efficiency of this portfolio following intravenous and intramuscular administration highlighted a clade-specific tropism. These studies further the design and selection of AAV capsids for gene therapy applications.

Economized large‐scale production of high yield of rAAV for gene therapy applications exploiting baculovirus expression system

The versatility of recombinant adeno-associated vector (rAAV) as a gene delivery system is due to the vector's ability to transduce different cell types as well as dividing and non-dividing cells. Large-scale production of rAAV remains one of the major challenges for continued development of pre-clinical and clinical studies, and for its potential commercialization. The baculovirus expression vectors (BEVS) and insect cells represent a potential method to produce rAAV economically at large scale. This technology uses three different BEVs (Bac-Rep, Bac-GFP, and Bac-VP) each at a multiplicity of infection (MOI) of 3. We reported previously the production of rAAV at 40 L scale using a stirred-tank bioreactor (STB). However, production in larger volumes is limited by the stability of the BEVs and amount of BEVs needed to achieve the target MOI of 3 per BEV. Here, the production parameters were optimized and the baculovirus stability was determined. The stability of the three types of baculovirus used to produce rAAV was determined for six expansion passages by protein expression analysis. To economize baculovirus, MOI and cell density at time of infection (TOI) were evaluated initially at small scale and then applied to the 10 L scale. An MOI = 0.03 and TOI cell density of 1 x 10(6) cells/mL produced high titer rAAV without comprising yield. To confirm the scalability of the process, rAAV was produced in a 10 L STB using the optimized parameters obtaining a 10x increase in yield ( approximately 1 x 10(14) rAAV DNAse-resistant particles per liter). These findings contribute to the process development for large-scale production of rAAV for gene therapy applications and its commercialization.

Adenovirus‐mediated small hairpin RNA targeting Bcl‐XL as therapy for colon cancer

Bcl-XL, an anti-apoptotic protein of Bcl-2 family, is overexpressed in colon cancers. To determine Bcl-XL's potential feasibility as a therapeutic target, we constructed a recombinant adenovirus that expressed a U6 promoter-driven small hairpin RNA (shRNA) targeting Bcl-XL (Ad/Bcl-XL shRNA) and evaluated the vector's ability to induce RNA interference in vivo and alter apoptosis induction in colon cancer cells and tumours. Ad/Bcl-XL shRNA effectively knocked down Bcl-XL expression in colon cancer cells and decreased their viability. Treatment with Ad/Bcl-XL shRNA but not control vectors led to dramatically increased cleavage of cellular apoptosis-related enzymes caspase-9, caspase-3 and poly(ADP-ribose) polymerase. Ad/Bcl-XL shRNA also significantly suppressed the growth of subcutaneous tumours derived from DLD1 cells in a nude mouse model and did so without causing any obvious damage to normal tissues or normal human fibroblasts. Together, our results support the feasibility of using adenovirus-mediated RNA interference therapy targeting Bcl-XL against colon cancers and warrant further studies of its safety and efficacy.

Coxsackie and Adenovirus Receptor Binding Ablation Reduces Adenovirus Liver Tropism and Toxicity

Human adenovirus-based vectors have emerged as a new promising vehicle for in vivo gene transfer-mediated therapy. However, the full potential of this methodology has not been fully realized because of the nonspecific tissue distribution of adenoviral vectors. Adenovirus infection is initiated by forming a complex between the fiber protein and a ubiquitously expressed host cell membrane protein, coxsackie B virus and adenovirus receptor (CAR). Therefore, ablating the adenovirus vector's ability to bind to the CAR is the first step in redirecting adenoviral tropism. To ablate CAR binding, we mutated the Bbeta sheet of the fiber knob, generating CAR-binding ablated replication-incompetent (dl-K420A-Z) and replication-competent (YKLK420A) adenoviral vectors. The in vitro transduction efficiency of dl-K420A-Z was significantly reduced in comparison to dl-LacZ carrying the wild-type fiber in CAR-positive cells but not in CAR-negative cells, suggesting that the mutation introduced in the Bbeta sheet of the fiber knob could disable the CAR-dependent transduction pathway. The in vivo transduction was also dramatically reduced in the liver and other organs for mice treated with dl-K420A-Z, compared with a cognate control vector, dl-LacZ. Concomitant with this attenuated gene transfer efficiency in vivo was a substantial reduction in the amount of general toxicity observed in the YKL-K420A-treated mice. Diminished toxicity was surmised from quantitative measurement of serum level of enzymes for liver and kidney function, hematologic chemistries, histopathology, and differences in lethality. Significant decrease in serum transaminases (alanine transferase [ALT] and aspartate transferase [AST]) was observed in mice treated with YKL-K420A. In addition, the lethality was lower in the YKLK420A- treated groups compared to the YKL-1-treated groups at all doses examined. Furthermore, the hepatopathologic analysis revealed that YKL-1 induced focal zonal necrosis and hepatocyte degeneration, while YKL-K420A induced mild spotty necrosis. In summary, this decreased vector tropism of CAR-binding ablated adenoviruses in normal tissues may increase the amount of virus available for infecting tumor cells and thus increase the antitumor efficacy with fewer unwanted side effects.

Gene Transfer in Hematopoietic Progenitor Cells Mediated by an SV40 Based Episomal Vector Carrying A S/MAR Element.

Background/Aims: Replicating episomal vectors present a potential alternative to currently used oncoretroviral vectors for gene transfer in hematopoietic progenitor/stem cells. Their main advantage is that they can persist in the recipient nucleus as independent units, without integrating into the host's genome, eliminating thus the risk of insertional mutagenesis. In the present study we explored the capacity of a recently developed SV40-based episomal vector, pEPI-eGFP, to stably transfect hematopoietic progenitor cell lines and primary cells, in order to evaluate its potential for therapeutic applications. pEPI-eGFP contains the enhanced green fluorescent protein (eGFP) cDNA and a Scaffold/Matrix Attachment Region (S/MAR) and it does not code for any proteins of viral origin. These unique properties qualify pEPI-eGFP as an attractive vehicle for gene therapy applications.Results: The vector was maintained as a stable episome in K562 cells for at least 100 generations and supported long-term EGFP expression, even in cells cultured in non-selective medium. Methylation-dependent cleavage assays demonstrated the vector's ability to self-replicate in K562 cells and MEL cells, whereas its episomal status was confirmed by Southern blotting and plasmid rescue in E. coli. The vector was also maintained in primary human fibroblasts for at least 30 passages with and without selection pressure.Transfection of CD34+ cells from umbilical cord blood with pEPI-eGFP was feasible by electroporation with an efficiency of up to 30%, as estimated by flow cytometric evaluation of eGFP expression 24–48 h post-transfection. Cytokine prestimulation of CD34+ cells did not enhance transfection efficiency, whereas transfection of post-mitotic cells, such as dendritic cells was also feasible. This suggests that efficient transfection with the vector does not require cell cycling. PCR with eGFP-specific primers in single CFU colonies derived from CD34+/eGFP+/PI− FACS-sorted cells demonstrated the presence of the vector in ~20% of the colonies.Conclusion: Our results demonstrate that pEPI-2 exhibits a considerable potential as a gene transfer vector for hematopoietic cells, as it confers long-term transgene expression in hematopoietic progenitor cell lines as a stable episome and can efficiently transfect unstimulated CD34+ cells. Further studies, both in vitro and in vivo, are required to assess the long-term maintenance in hematopoietic progenitor/stem cells and their progeny as well as other features of the vector, in order to evaluate its overall efficacy and possibly improve its performance.

126. Zc-Ad Vector: A Novel Immunotargeting Vector with a Modified Fc-Binding Domain

Adenovirus serotype 5 (Ad5) has shown potential as a gene delivery vehicle for numerous gene therapy applications. Because of the paucity of Ad5's primary receptor, coxsackie-adenovirus receptor (CAR), on cancer cells, and its widespread distribution on other cell types, vector targeting to specific receptors on cancer cells has become a mandate for efficient gene transfer. In one targeting scheme, the receptor-selective affinity of immunoglobin G (IgG) molecules has been employed to retarget Ad5 to specific cell types. This system provides for a flexible, modular approach to targeting cancer cells, because of the vector's ability to utilize any of the multiple IgG molecules with characterized receptor affinity. In this targeting schema, the Ad5 fiber capsid protein has been engineered to contain a specific domain (Cd) of protein A from S. aureus, exhibiting binding affinity for the conserved Fc domain common to all IgG molecules. Attempts to fully develop this technology, however, have been confounded by high degrees of viral aggregation, observed after viral-IgG complexes have been formed ex vivo, thus hindering the vector's ability to transfer genes. The proposed cause of this aggregation is the Cd domain's ability to bind the Fab regions of the IgG molecule, in addition to the Fc domain, yielding complexes of multiple virions crosslinked via IgG molecules. To circumvent this problem, we have engineered a novel IgG binding ligand, the Zc domain; a modification of the previous Fc-binding domain employed for this targeting schema. Based on the literature of well-known non-Fab-binding, Fc-binding domains, we have abolished the Zc domain's ability to bind the Fab regions of IgG molecules, via site-directed mutagenesis of a single glycine to alanine substitution. With this structural modification, we hypothesize that the degree of aggregation displayed in Ad-Ig complexes will be lessened, thus, enhancing the vector's ability to transfer genes in situ. Given the promise shown for modifications of this nature at the C-terminus, we have constructed an E1-deleted Ad5 vector containing a C-terminal linker with the Zc domain, and enhanced green fluorescent protein (GFP) as a reporter gene. The Zc-Ad vectors were propagated and incubated with either a monoclonal anti-CD40 IgG, or an Fc anti-CD40 single chain antibody (scFv) fusion protein to provide for CD40 targeted Ad5 vectors. Gene transfer analyses, comparing the targeting efficacy of the Zc vs. Cd viruses, complexed with the IgG and scFv fusion, were carried out with 293 cells expressing CD40. Preliminary data for these experiments demonstrate that the Zc-Ad vector is equally effective as our previous Ig-binding vector system, as displayed by CAR-independent, CD40-mediated gene transfer in vitro. Because aggregation has apparently undermined the potential utility of targeting by Ig-binding Ads hitherto, we propose that the Zc ligand will mitigate aggregation, and optimize these vectors for efficient gene transfer.

A novel approach to gene therapy of albino hair in histoculture with a retroviral streptomyces tyrosinase gene.

In order to induce melanin production in mammalian cells with pigment disorders such as albino hair, a recombinant retrovirus containing the mel locus of Streptomyces antibioticus was constructed. The S. antibioticus mel locus, which consists of the open reading frame (ORF)-438 and the tyrosinase gene, was specifically derived by polymerase chain reaction (PCR) from Streptomyces plasmid pIJ702. The ORF-438 is required for the transfer of copper to apotyrosinase, which is essential for tyrosinase enzymatic activity. The tyrosinase gene was inserted into the XhoI/BamHI cloning site of the pLXSN retroviral vector to obtain pLtyrSN. An internal ribosome entry site (IRES) suitable for mammalian cell expression was obtained from the pLXIN retroviral vector by PCR. The ORF-438 and IRES DNA fragments were inserted into the pLtyrSN vector to obtain the tyrosinase-expression retroviral vector pLmelSN. The expression vector was amplified in murine PT67 packaging cells, where the ORF-438 and tyrosinase genes were also co-expressed as determined by reverse transcription-PCR. In order to evaluate the vector's ability to restore pigment production in cells with a pigment disorder, albino-mouse skins were histocultured and then infected with the pLmelSN retrovirus. Six days after infection, melanin granules were observed in approximately 60% of albino-mouse hair follicles in the histocultured skin. These results demonstrated that the S. antibioticus mel operon could express an active tyrosinase and produce melanin in the albino-mouse hair follicles. This novel gene therapy approach, using a small and simple tyrosinase operon in a high-expression vector, has a potentially wide application for therapy of pigment disorders in hair follicles.

Effective gene transfer into regenerating sciatic nerves by adenoviral vectors: potentials for gene therapy of peripheral nerve injury.

Replication defective adenoviral vectors have been demonstrated as an effective method for delivering genes into a variety of cell types and tissues both in vivo and in vitro. Transfecting genes into neuronal cells has proven to be difficult because of their lack of cell division. Since the major problem in neurological disease is the degeneration of the terminally differentiated neuronal cells, the adenoviral vector's ability to transfer genes into differentiated post-mitotic cells makes them advantageous for a gene delivery system for the nervous system. Here we showed that a replication defective recombinant adenovirus carrying the lacZ gene could infect the neuronal stem cells and even the differentiated neuronal cells derived from the central nervous system. The lacZ gene delivered into the neuronal cells was expressed efficiently. In addition, the recombinant virus also infected Schwann cells in intact and injured nerves in vivo. The expression of the lacZ gene lasted for 5 weeks, within which nerve regeneration is accomplished in the rat. Adenoviral vectors might thus be used to modulate Schwann cell gene expression for treating peripheral nerve injury or peripheral neuropathy.