Feline Immunodeficiency Virus Vectors Research Articles

Intrapulmonary Versus Nasal Transduction of Murine Airways With GP64-pseudotyped Viral Vectors

Persistent viral vector-mediated transgene expression in the airways requires delivery to cells with progenitor capacity and avoidance of immune responses. Previously, we observed that GP64-pseudotyped feline immunodeficiency virus (FIV)-mediated gene transfer was more efficient in the nasal airways than the large airways of the murine lung. We hypothesized that in vivo gene transfer was limited by immunological and physiological barriers in the murine intrapulmonary airways. Here, we systematically investigate multiple potential barriers to lentiviral gene transfer in the airways of mice. We show that GP64-FIV vector transduced primary cultures of well-differentiated murine nasal epithelia with greater efficiency than primary cultures of murine tracheal epithelia. We further demonstrate that neutrophils, type I interferon (IFN) responses, as well as T and B lymphocytes are not the major factors limiting the transduction of murine conducting airways. In addition, we observed better transduction of GP64-pseudotyped vesicular stomatitis virus (VSV) in the nasal epithelia compared with the intrapulmonary airways in mice. VSVG glycoprotein pseudotyped VSV transduced intrapulmonary epithelia with similar efficiency as nasal epithelia. Our results suggest that the differential transduction efficiency of nasal versus intrapulmonary airways by FIV vector is not a result of immunological barriers or surface area, but rather differential expression of cellular factors specific for FIV vector transduction.

Gene Transfer to Chicks Using Lentiviral Vectors Administered via the Embryonic Chorioallantoic Membrane

The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV), into the chorioallantoic membrane (CAM) of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP) or recombinant alpha-melanocyte-stimulating hormone (α-MSH) genes, driven by the cytomegalovirus (CMV) promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ∼0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1)-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA), and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides.

A Lentiviral Vector-Based, Herpes Simplex Virus 1 (HSV-1) Glycoprotein B Vaccine Affords Cross-Protection against HSV-1 and HSV-2 Genital Infections

Genital herpes is caused by herpes simplex virus 1 (HSV-1) and HSV-2, and its incidence is constantly increasing in the human population. Regardless of the clinical manifestation, HSV-1 and HSV-2 infections are highly transmissible to sexual partners and enhance susceptibility to other sexually transmitted infections. An effective vaccine is not yet available. Here, HSV-1 glycoprotein B (gB1) was delivered by a feline immunodeficiency virus (FIV) vector and tested against HSV-1 and HSV-2 vaginal challenges in C57BL/6 mice. The gB1 vaccine elicited cross-neutralizing antibodies and cell-mediated responses that protected 100 and 75% animals from HSV-1- and HSV-2-associated severe disease, respectively. Two of the eight fully protected vaccinees underwent subclinical HSV-2 infection, as demonstrated by deep immunosuppression and other analyses. Finally, vaccination prevented death in 83% of the animals challenged with a HSV-2 dose that killed 78 and 100% naive and mock-vaccinated controls, respectively. Since this FIV vector can accommodate two or more HSV immunogens, this vaccine has ample potential for improvement and may become a candidate for the development of a truly effective vaccine against genital herpes.

Transcriptional Targeting in the Airway Using Novel Gene Regulatory Elements

The delivery of cystic fibrosis transmembrane conductance regulator (CFTR) to airway epithelia is a goal of many gene therapy strategies to treat cystic fibrosis. Because the native regulatory elements of the CFTR are not well characterized, the development of vectors with heterologous promoters of varying strengths and specificity would aid in our selection of optimal reagents for the appropriate expression of the vector-delivered CFTR gene. Here we contrasted the performance of several novel gene-regulatory elements. Based on airway expression analysis, we selected putative regulatory elements from BPIFA1 and WDR65 to investigate. In addition, we selected a human CFTR promoter region (∼ 2 kb upstream of the human CFTR transcription start site) to study. Using feline immunodeficiency virus vectors containing the candidate elements driving firefly luciferase, we transduced murine nasal epithelia in vivo. Luciferase expression persisted for 30 weeks, which was the duration of the experiment. Furthermore, when the nasal epithelium was ablated using the detergent polidocanol, the mice showed a transient loss of luciferase expression that returned 2 weeks after administration, suggesting that our vectors transduced a progenitor cell population. Importantly, the hWDR65 element drove sufficient CFTR expression to correct the anion transport defect in CFTR-null epithelia. These results will guide the development of optimal vectors for sufficient, sustained CFTR expression in airway epithelia.

Lentiviral Vector Gene Transfer to Porcine Airways

In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1–based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF).

Feline Immunodeficiency Virus-Based Lentiviral Vectors: Figure 1.

Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This article describes the production and use of advanced generation FIV vectors. Key properties are discussed in comparison to other lentiviral vectors. Additional topics include the practical implications of species-specific retroviral restriction factors and the production of nonintegrating FIV vectors.

Altering α-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism.

BackgroundThe envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV) can efficiently pseudotype lentiviral vectors. Some strains of LCMV exploit high affinity interactions with α-dystroglycan (α-DG) to bind to cell surfaces and subsequently fuse in low pH endosomes. LCMV strains with low α-DG affinity utilize an unknown receptor and display unique tissue tropisms. We pseudotyped non-primate feline immunodeficiency virus (FIV) vectors using LCMV derived glycoproteins with high or low affinity to α-DG and evaluated their properties in vitro and in vivo.MethodsWe pseudotyped FIV with the LCMV WE54 strain envelope glycoprotein and also engineered a point mutation in the WE54 envelope glycoprotein (L260F) to diminish α-DG affinity and direct binding to alternate receptors. We hypothesized that this change would alter in vivo tissue tropism and enhance gene transfer to neonatal animals.ResultsIn mice, hepatic α- and β-DG expression was greatest at the late gestational and neonatal time points. When displayed on the surface of the FIV lentivirus the WE54 L260F mutant glycoprotein bound weakly to immobilized α-DG. Additionally, LCMV WE54 pseudotyped FIV vector transduction was neutralized by pre-incubation with soluble α-DG, while the mutant glycoprotein pseudotyped vector was not. In vivo gene transfer in adult mice with either envelope yielded low transduction efficiencies in hepatocytes following intravenous delivery. In marked contrast, neonatal gene transfer with the LCMV envelopes, and notably with the FIV-L260F vector, conferred abundant liver and lower level cardiomyocyte transduction as detected by luciferase assays, bioluminescent imaging, and β-galactosidase staining.ConclusionsThese results suggest that a developmentally regulated receptor for LCMV is expressed abundantly in neonatal mice. LCMV pseudotyped vectors may have applications for neonatal gene transfer.AbbreviationsArmstrong 53b (Arm53b); baculovirus Autographa californica GP64 (GP64); charge-coupled device (CCD); dystroglycan (DG); feline immunodeficiency virus (FIV); glycoprotein precursor (GP-C); firefly luciferase (Luc); lymphocytic choriomeningitis virus (LCMV); nuclear targeted β-galactosidase (ntLacZ); optical density (OD); PBS/0.1% (w/v) Tween-20 (PBST); relative light units (RLU); Rous sarcoma virus (RSV); transducing units per milliliter (TU/ml); vesicular stomatitis virus (VSV-G); wheat germ agglutinin (WGA); 50% reduction in binding (C50).

MiR-301a as an NF-κB activator in pancreatic cancer cells

NF-κB is constitutively activated in most human pancreatic adenocarcinoma, which is a deadly malignancy with a 5-year survival rate of about 5%. In this work, we investigate whether microRNAs (miRNAs) contribute to NF-κB activation in pancreatic cancer. We demonstrate that miR-301a down-regulates NF-κB-repressing factor (Nkrf) and elevates NF-κB activation. As NF-κB promotes the transcription of miR-301a, our results support a positive feedback loop as a mechanism for persistent NF-κB activation, in which miR-301a represses Nkrf to elevate NF-κB activity, which in turn promotes miR-301a transcription. Nkrf was found down-regulated and miR-301a up-regulated in human pancreatic adenocarcinoma tissues. Moreover, miR-301a inhibition or Nkrf up-regulation in pancreatic cancer cells led to reduced NF-κB target gene expression and attenuated xenograft tumour growth, indicating that miR-301a overexpression contributes to NF-κB activation. Revealing this novel mechanism of NF-κB activation by an miRNA offers new avenues for therapeutic interventions against pancreatic cancer.

Neonatal Gene Therapy of Glycogen Storage Disease Type Ia Using a Feline Immunodeficiency Virus–based Vector

Glycogen storage disease type Ia (GSD-Ia), also known as von Gierke disease, is caused by a deficiency of glucose-6-phosphatase-alpha (G6Pase), a key enzyme in glucose homeostasis. From birth, affected individuals cannot maintain normal blood glucose levels and suffer from a variety of metabolic disorders, leading to life-threatening complications. Gene therapy has been proposed as a possible option for treatment of this illness. Vectors have been constructed from feline immunodeficiency virus (FIV), a nonprimate lentivirus, because the wild-type virus does not cause disease in humans. Previously, we have shown that these vectors are capable of integrating stably into hepatocyte cell lines and adult murine livers and lead to long-term transgene expression. In the current work, we have assessed the ability to attenuate disease symptoms in a murine model of GSD-Ia. Single administration of FIV vectors containing the human G6Pase gene to G6Pase-alpha(-/-) mice did not change the biochemical and pathological phenotype. However, a double neonatal administration protocol led to normalized blood glucose levels, significantly extended survival, improved body weight, and decreased accumulation of liver glycogen associated with the disease. This approach shows a promising paradigm for treating GSD-Ia patients early in life thereby avoiding long-term consequences.

Feline immunodeficiency virus vector as a tool for preventative strategies against human breast cancer

Breast cancer is the most common type of neoplasia in women. Currently 90% cases are sporadic whereas up to 10% are hereditary and likely due to germ-line mutations in specific genes. Eighty percent hereditary breast cancers are associated with inactivation of breast cancer-associated genes (BRCA) type 1 and 2 by sequential mutations. Loss of functionality of one or both genes greatly increases the risk to develop breast cancer and set the basis for the design of strategies to restore BRCA functions or replace the inactive gene(s) before the emergence of the neoplasia. We have produced a lentiviral vector from the feline immunodeficiency virus (FIV) to transduce wild type BRCA1 into primary mammary cells with a non-functional gene. The system was set up and optimized in tumor cells expressing a truncated gene. Transduced BRCA1 was expressed efficiently and fully functional as demonstrated by the restored ability to repair DNA damages upon exposure of transduced cells to ionizing radiations. This work sets the basis for innovative gene therapy strategies against human breast cancer.

Spinal interleukin‐1β in a mouse model of arthritis and joint pain

Pain from arthritis has been associated with peripheral sensitization of primary sensory afferents and the development of inflammation at the dorsal horns. This study was undertaken to determine whether the role of spinal interleukin-1beta (IL-1beta) in central processing of pain is important in the development of arthritis. Col1-IL-1betaXAT mice and GFAP-IL-1betaXAT mice were injected with the feline immunodeficiency virus (FIV) (Cre) vector in the right and left temporomandibular joints (TMJs), or in the cisterna magna, respectively, to induce IL-1beta expression in the dorsal horns of the spinal horn. To inhibit intrathecal IL-1 receptor type I (IL-1RI) signaling, FIV(IL-1Ra) vector was injected into the cisterna magna of Col1-IL-1betaXAT mice. The effects of IL-1RI receptor inhibition in GFAP-IL-1betaXAT mice were studied in the GFAP-IL-1betaXAT-IL-1RI(-/-) compound mouse model. Neuroinflammatory, sensory, and behavioral changes were evaluated in conjunction with arthritic changes in the TMJ, assessed by histopathologic and immunohistochemical analyses. Induction of an osteoarthritis-like condition in the TMJ in the Col1-IL-1betaXAT mouse model resulted in up-regulation of murine IL-1beta at the dorsal horns. Moreover, intrathecal inhibition of IL-1RI in Col1-IL-1betaXAT mice with arthritis led to amelioration of joint pathology and attenuation of the attendant joint pain. Overexpression of spinal IL-1beta in the recently developed GFAP-IL-1betaXAT somatic mosaic model of neuroinflammation led to development of arthritis-like pathology accompanied by increased pain-like behavior. Our results indicate that joint pathology and pain are dependent on spinal IL-1beta, and suggest the presence of a bidirectional central nervous system-peripheral joints crosstalk that may contribute to the development, expansion, and exacerbation of arthritis.

Expression of truncated dystrophin cDNAs mediated by a lentiviral vector

The success of Duchenne muscular dystrophy gene therapy requires promising tools for gene delivery and mini-gene cassettes that can express therapeutic levels of a functional protein. To explore the expression feasibility of truncated dystrophin cDNAs mediated by a lentiviral vector derived from feline immunodeficiency virus. Three truncated dystrophin cDNAs were constructed by PCR cloning, then these cDNAs were inserted into lentiviral vectors. Recombinant lentiviruses were generated by transient transfection of lentiviral vector constructs into 293Ad5+ cells. Cultured myoblasts were then infected with recombinant lentiviruses. Expression of truncated dystrophin cDNAs was detected by Western blot analysis. Mediated by lentiviral vectors, three cDNAs constructed by PCR cloning expressed relative truncated dystrophins in cultured myoblasts. Truncated dystrophin cDNAs can express themselves successfully mediated by feline immunodeficiency virus vectors. It offers the possibility of an approach utilizing truncated dystrophin cDNAs and lentiviral vectors toward gene therapy of Duchenne muscular dystrophy.

Amelioration of pain and histopathologic joint abnormalities in the Col1‐IL‐1βXAT mouse model of arthritis by intraarticular induction of μ‐opioid receptor into the temporomandibular joint

To evaluate opioid receptor function as a basis for novel antinociceptive therapy in arthritis. We induced human mu-opioid receptor (HuMOR) expression in arthritic joints of mice, using the feline immunodeficiency virus (FIV) vector, which is capable of stably transducing dividing, growth-arrested, and terminally differentiated cells. Male and female Col1-IL-1beta(XAT)-transgenic mice developed on a C57BL/6J background and wild-type littermates were studied. A single injection of FIV(HuMOR) into the temporomandibular joints of Col1-IL-1beta(XAT)-transgenic mice 1 week prior to induction of arthritis prevented the development of orofacial pain and joint dysfunction, and reduced the degree of histopathologic abnormality in the joint. In addition, FIV(HuMOR) prevented the attendant sensitization of trigeminal sensory neurons and activation of astroglia in brainstem trigeminal sensory nuclei. These effects were mediated by the transduction of primary sensory neurons via transport of FIV vectors from peripheral nerve endings to sensory ganglia, as evidenced by HuMOR expression in neuronal cell bodies located in the trigeminal ganglia, as well as in their proximal and distal nerve branches located in the main sensory and subnucleus caudalis of the brainstem and joints, respectively. The presence of MOR ligands predominantly in the descending trigeminal nucleus suggested that the observed antinociception occurred at the subnucleus caudalis. Articular chondrocytes and meniscal tissue were also infected by FIV(HuMOR), which presumably exerted an antiinflammatory effect on cartilage. Our results indicate that prophylactic therapy with MOR overexpression in joints can successfully prevent the development of pain, dysfunction, and histopathologic abnormalities in the joints in arthritis. These findings may provide a basis for the future development of spatiotemporally controlled antinociceptive and antiinflammatory therapy for arthritis.

Streamlined design of a self-inactivating feline immunodeficiency virus vector for transducing ex vivo dendritic cells and T lymphocytes

BackgroundSafe and efficient vector systems for delivering antigens or immunomodulatory molecules to dendritic cells (DCs), T lymphocytes or both are considered effective means of eliciting adaptive immune responses and modulating their type, extent, and duration. As a possible tool toward this end, we have developed a self-inactivating vector derived from feline immunodeficiency virus (FIV) showing performance characteristics similar to human immunodeficiency virus-derived vectors but devoid of the safety concerns these vectors have raised.MethodsThe pseudotyped FIV particles were generated with a three-plasmid system consisting of: the packaging construct, providing Gag, Pol and the accessory proteins; the vector(s), basically containing FIV packaging signal (ψ), Rev responsive element, R-U5 region at both ends, and the green fluorescent protein as reporter gene; and the Env plasmid, encoding the G protein of vesicular stomatitis virus (VSV-G) or the chimeric RD114 protein. Both packaging and vector constructs were derived from p34TF10, a replication competent molecular clone of FIV. The pseudotyped particles were produced by transient transfection in the Crandell feline fibroblast kidney (CrFK) or the human epithelial (293T) cell line.ResultsTo broaden its species tropism, the final vector construct was achieved through a series of intermediate constructs bearing a longer ψ, the FIV central polypurin tract sequence (cPPT), or the woodchuck hepatitis post-regulatory element (WPRE). These constructs were compared for efficiency and duration of transduction in CrFK or 293T cells and in the murine fibroblast cell line NIH-3T3. Whereas ψ elongation and cPPT addition did not bring any obvious benefit, insertion of WPRE downstream GFP greatly improved vector performances. To maximize the efficiency of transduction for ex-vivo murine DCs and T-lymphocytes, this construct was tested with VSV-G or RD114 and using different transduction protocols. The results indicated that the FIV construct derived herein stably transduced both cell types, provided that appropriate vector makeup and transduction protocol were used. Further, transduced DCs underwent changes suggestive of an induced maturation.ConclusionIn contrast to previously described FIV vectors that were poorly efficient in delivering genetic material to DCs and T lymphocytes, the vector developed herein has potential for use in experimental immunization strategies.

Optimization of Feline Immunodeficiency Virus Vectors for RNA Interference

RNA interference (RNAi) occurs naturally in plant and animal cells as a means for modulating gene expression. This process has been experimentally manipulated to achieve targeted gene silencing in cells, tissues, and animals, using a variety of vector systems. Here, we tested the hypothesis that vectors based on feline immunodeficiency virus (FIV) could be used for coexpression of reporter constructs and RNAi expression cassettes. We found, unexpectedly, in our initial constructs that placement of RNAi expression cassettes downstream from a polymerase II (pol II)-expressed reporter gene inhibited reporter expression but not vector titer. Through a series of intermediate vector constructs, we found that placement of the RNAi expression cassette relative to the Rev response element and the pol II expression cassette was critical for efficient RNAi and reporter gene expression. These results suggested that steric factors, including RNA structure and recruitment of competing transcriptional machinery, may affect gene expression from FIV vectors. In a second series of studies, we show that target sequence silencing can be achieved in cells transduced by FIV vectors coexpressing reporter genes and 3' untranslated region resident microRNAs. The optimized FIV-based RNAi expression vectors will find broad use given the extensive tropism of pseudotyped FIV vectors for many cell types in vitro and in vivo.

371. Optimization of Feline Immunodeficiency Viral Vectors for RNA Interference

RNA interference (RNAi) refers to post-transcriptional reduction of cellular gene expression mediated by sequence specific, 21 nucleotide double-stranded RNA molecules. RNAi occurs naturally in mammals (microRNAs), and appears to be particularly involved in controlling development of the nervous system. In addition, RNAi can be induced through exogenous expression of short hairpin RNAs (shRNA) using viral vectors. Feline immunodeficiency virus (FIV)- based lentiviral vectors are useful tools for neuronal gene delivery because they can stably infect difficult-to-transduce cells, are easy to produce, and, unlike the more broadly utilized HIV-based vectors, are non-human pathogens that pose a lower risk of generating replication-competent viruses in humans. Despite these advantages, compared to HIV vectors, less is known about the biology and construction of FIV and FIV-based vectors. Here, we describe thedevelopment of a second-generation FIV-based vector system that expresses U6 promoter-transcribed shRNAs (shLacZ) and a GFP reporter gene from the same vector. We found that the position of the U6 expression cassette with respect to the GFP reporter and viral rev-responsive element (RRE) was critical for reporter gene and shRNA expression. In particular, placement of the U6 promoter alone or an U6.shLacZ cassette near the 3' end of the vector genome between the GFP reporter cassette and the viral RRE caused 31 and 131-fold reductions, respectively, in transducing unit titer compared to an FIV vector expressing GFP alone. Reduced titer was also observed when a similarly-organized RNA pol III promoter (H1) was cloned into the same site. In contrast, TU titer was slightly increased (2.8-fold) when a similarly-sized, non-coding fragment of firefly luciferase was cloned into the same site downstream of GFP. Movement of U6.shLacZ to a site upstream of GFP resulted in complete TU titer restoration. Taken together, these data demonstrate that GFP expression is inhibited when a pol III-based promoter is located downstream of the reporter, suggesting that this inhibition may depend on recruitment of pol III transcription machinery.

Perfusion of His-Tagged Eukaryotic Myocilin Increases Outflow Resistance in Human Anterior Segments in the Presence of Aqueous Humor

A previous study by the authors has shown that recombinant myocilin purified from a prokaryotic expression system increases outflow resistance in cultured human anterior segments. The present study was performed to determine whether full-length myocilin purified from a human trabecular meshwork cell expression system alters outflow resistance after infusion into human anterior segments. A feline immunodeficiency virus vector encoding both full-length myocilin (amino acids 1-503 fused to C-terminal V5 and six-histidine epitopes) and puromycin resistance was used to transduce a transformed trabecular meshwork cell line (TM5). Stably expressing cells were selected with puromycin. Recombinant myocilin was purified from the media using nickel ion affinity chromatography. Control purifications were performed on media from parental TM5 cells. Anterior segments of human eyes were placed in organ culture and perfused with either Dulbecco's modified Eagle's medium (DMEM) or DMEM supplemented with 50% porcine aqueous humor. One eye received an anterior chamber exchange with recombinant myocilin (2 microg/mL), whereas the fellow eye received an equal volume of control. Immunohistochemistry was performed with anti-myocilin and anti-V5 antibodies. Native polyacrylamide gel electrophoresis was used to analyze myocilin complex formation in porcine aqueous humor. Recombinant myocilin in porcine aqueous humor increased outflow resistance in cultured human anterior segments (91% +/- 68% [mean +/- SD] versus 18% +/- 31% in fellow control eye; n = 9, P = 0.004). Maximum outflow resistance was obtained 5 to 17 hours after infusion and remained above baseline for >3 days. Recombinant myocilin also increased outflow resistance in eyes incubated in DMEM, but only if myocilin was preincubated with porcine aqueous humor (78% +/- 77% when preincubated in DMEM containing porcine aqueous humor versus 13% +/- 15% when preincubated with DMEM alone, n = 6, P = 0.03). Recombinant myocilin appears to form a complex in porcine aqueous humor with a heat-labile protein(s). Immunohistochemistry revealed the presence of myocilin in the juxtacanalicular region of the trabecular meshwork. Myocilin purified from human trabecular meshwork cells increased outflow resistance in cultured human anterior segments, but only after incubation with porcine aqueous humor. Recombinant myocilin appears to form a complex in porcine aqueous humor that enables it to bind specifically within the trabecular meshwork.

Restriction of feline immunodeficiency virus by Ref1, Lv1, and primate TRIM5alpha proteins.

The Ref1 and Lv1 postentry restrictions in human and monkey cells have been analyzed for lentiviruses in the primate and ungulate groups, but no data exist for the third (feline) group. We compared feline immunodeficiency virus (FIV) to other restricted (human immunodeficiency virus type 1 [HIV-1], equine infectious anemia virus [EIAV]) and unrestricted (NB-tropic murine leukemia virus [NB-MLV]) retroviruses across wide ranges of viral inputs in cells from multiple primate and nonprimate species. We also characterized restrictions conferred to permissive feline and canine cells engineered to express rhesus and human TRIM5alpha proteins and performed RNA interference (RNAi) against endogenous TRIM5alpha. We find that expression of rhesus or human TRIM5alpha proteins in feline cells restricts FIV, impairing pseudotyped vector transduction and viral replication, but rhesus TRIM5alpha is more restricting than human TRIM5alpha. Notably, however, canine cells did not support restriction by human TRIM5alpha and supported minimal restriction by rhesus TRIM5alpha, suggesting that these proteins may not function autonomously or that a canine factor interferes. Stable RNAi knockdown of endogenous rhesus TRIM5alpha resulted in marked increases in FIV and HIV-1 infectivities while having no effect on NB-MLV. A panel of nonprimate cell lines varied widely in susceptibility to lentiviral vector transduction, but normalized FIV and HIV-1 vectors varied concordantly. In contrast, in human and monkey cells, relative restriction of FIV compared to HIV-1 varied from none to substantial, with the greatest relative infectivity deficit for FIV vectors observed in human T-cell lines. Endogenous and introduced TRIM5alpha restrictions of FIV could be titrated by coinfections with FIV, HIV-1, or EIAV virus-like particles. Arsenic trioxide had complex and TRIM5alpha-independent enhancing effects on lentiviral but not NB-MLV infection. Implications for human gene therapy are discussed.

Persistent expression of factor VIII in vivo following nonprimate lentiviral gene transfer

AbstractHemophilia A is a clinically important coagulation disorder caused by the lack or abnormality of plasma coagulation factor VIII (FVIII). Gene transfer of the FVIII cDNA to hepatocytes using lentiviral vectors is a potential therapeutic approach. We investigated the efficacy of feline immunodeficiency virus (FIV)–based vectors in targeting hepatocytes and correcting FVIII deficiency in a hemophilia A mouse model. Several viral envelope glycoproteins were screened for efficient FIV vector pseudotyping and hepatocyte transduction. The GP64 glycoprotein from baculovirus Autographa californica multinuclear polyhedrosis virus pseudo-typed FIV efficiently and showed excellent hepatocyte tropism. The GP64-pseudotyped vector was stable in the presence of human or mouse complement. Inclusion of a hybrid liver-specific promoter (murine albumin enhancer/human α1-antitrypsin promoter) further enhanced transgene expression in hepatocytes. We generated a GP64-pseudotyped FIV vector encoding the B domain–deleted human FVIII coding region driven by the liver-specific promoter, with 2 beneficial point mutations in the A1 domain. Intravenous vector administration conferred sustained FVIII expression in hemophilia A mice for several months without the generation of anti–human FVIII antibodies and resulted in partial phenotypic correction. These findings demonstrate the utility of GP64-pseudotyped FIV lentiviral vectors for targeting hepatocytes to correct disorders associated with deficiencies of secreted proteins.

Prolonged transgene expression in murine salivary glands following non-primate lentiviral vector transduction

Salivary glands are an accessible organ for gene therapy, enabling expression of recombinant proteins for both exocrine and endocrine secretion. Lentivirus-based vectors have many advantages for gene therapy, including their ability to infect nondividing cells and to stably integrate into the host genome, enabling long-term transgene expression without eliciting an inflammatory immune response. In the present study, murine salivary glands were inoculated with feline immunodeficiency virus (FIV)-based lentiviral vectors expressing various reporter genes. Luciferase expression was observed as early as 24 h posttransduction, peaked at 17-21 days, and remained stable for more than 80 days. Staining with X-gal suggested that mucous acinar cells were effectively transduced. FIV vector transduction with the secreted alkaline phosphatase gene increased serum levels in treated animals for up to 45 days, and the FIV vector harboring the interferon-gamma (IFN-gamma) expression cassette induced an increase in IFN-gamma serum levels as well as in the supernatant of salivary gland explant cultures. These results demonstrate that the transduction of salivary glands with nonprimate lentiviral vectors may provide a novel and highly effective vehicle for long-term endocrine transgene expression.