In vivo treatment of hemophilia A and mucopolysaccharidosis type VII using nonprimate lentiviral vectors.

Gene therapy holds great promise for the treatment of a variety of inherited diseases, including hemophilia A and mucopolysaccharidosis type VII (MPS VII). In both these disorders, subnormal levels of replacement protein have therapeutic effects. Thus we hypothesized that transduction of a small proportion of cells by feline immunodeficiency virus (FIV)-based lentiviral vectors might provide sufficient levels of transgene expression for phenotypic correction. We intravenously injected replication-deficient FIV-based vectors encoding either human factor VIII or human beta-glucuronidase into factor VIII-deficient or beta-glucuronidase-deficient mice, respectively. This route of delivery targeted multiple organs, with the liver as the primary transduction site. In the hemophilia A mice, factor VIII expression persisted for the duration of the experiments (approximately 5 months), and recipient mice survived an otherwise lethal bleeding episode (tail-clipping). In mucopolysaccharidosis type VII mice, substantial beta-glucuronidase activity was detected in several tissues and corresponded with marked reduction of lysosomal storage in liver and spleen. These findings indicate that gene transfer with FIV-based lentiviral vectors can permanently introduce transgenes into a sufficient number of hepatocytes for long-term therapeutic effect and suggest potential clinical value of FIV-based lentiviral vectors for treatment of hemophilia A and MPS VII.

Long-term reversal of hypercholesterolemia in low density lipoprotein receptor (LDLR)-deficient mice by adenovirus-mediated LDLR gene transfer combined with CD154 blockade.

BACKGROUND: Deficiency of the low density lipoprotein receptor (LDLR) results in abnormal elevation of cholesterol within the intermediate and low density plasma lipoproteins (IDL/LDL), and predisposes to early onset atherosclerosis. Cholesterol reduction after adenovirus-mediated LDLR gene transfer to LDLR-deficient animals is transient, due to the elicitation of an immune response. METHODS: The LDLR-deficient mouse was used as a model to investigate adenovirus-mediated LDLR gene transfer combined with short-term immunosuppression as a cholesterol lowering therapy. Mice were infused with replication-deficient recombinant adenovirus encoding LDLR under control of the cytomegalovirus promoter/enhancer (AdLDLR), and injected with a blocking antibody directed against CD154 (CD40 ligand) to suppress immune responses against the vector and foreign transgene product. RESULTS: Mice given AdLDLR and treated with anti-CD154 expressed LDLR on hepatocytes and maintained cholesterol levels below or within normal range for at least 92 days. In contrast, without adjunct immunosuppression LDLR expression was transient, corresponding to temporary decline in cholesterol levels. Analysis of cholesterol concentrations in fractionated plasma showed remarkable reduction in all lipoprotein fractions at early time-points after gene transfer. At later time-points, non-immunosuppressed control mice regained the disease profile with elevated IDL/LDL cholesterol, while profiles of anti-CD154-treated mice were similar to normal. LDLR mRNA transcripts were present in livers of the anti-CD154-treated mice but not controls, 93 days after AdLDLR injection. However, vector DNA was detected in livers of both groups. These results suggest that loss of LDLR expression in the non anti-CD154-treated mice was in part due to immune-mediated promoter silencing, and that anti-CD154 prevented this effect. CONCLUSION: Treatment with anti-CD154 antibody inhibits immune-mediated loss of transgene expression, enabling long-term reduction in cholesterol levels after AdLDLR gene transfer to LDLR-deficient mice.

Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system.

Recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in the central nervous system (CNS), but it is not known how other rAAV serotypes perform as CNS gene transfer vectors. Serotypes 4 and 5 are distinct from rAAV2 and from each other in their capsid regions, suggesting that they may direct binding and entry into different cell types. In this study, we examined the tropisms and transduction efficiencies of beta-galactosidase-encoding vectors made from rAAV4 and rAAV5 compared with similarly designed rAAV2-based vectors. Injection of rAAV5 beta-galactosidase (betagal) or rAAV4betagal into the lateral ventricle resulted in stable transduction of ependymal cells, with approximately 10-fold more positive cells than in mice injected with rAAV2betagal. Major differences between the three vectors were revealed upon striatal injections. Intrastriatal injection of rAAV4betagal resulted again in striking ependyma-specific expression of transgene, with a notable absence of transduced cells in the parenchyma. rAAV2betagal and rAAV5betagal intrastriatal injections led to beta-gal-positive parenchymal cells, but, unlike rAAV2betagal, rAAV5betagal transduced both neurons and astrocytes. The number of transgene-positive cells in rAAV5betagal-injected brains was 130 and 5,000 times higher than in rAAV2betagal-injected brains at 3 and 15 wk, respectively. Moreover, transgene-positive cells were widely dispersed throughout the injected hemisphere in rAAV5betagal-transduced animals. Together, our data provide in vivo support for earlier in vitro work, suggesting that rAAV4 and rAAV5 gain cell entry by means of receptors distinct from rAAV2. These differences could be exploited to improve gene therapy for CNS disorders.

Systemic and central nervous system correction of lysosomal storage in mucopolysaccharidosis type VII mice.

Mucopolysaccharidosis (MPS) type VII patients lack functional beta-glucuronidase, leading to systemic and central nervous system dysfunction. In this study we tested whether recombinant adenovirus that encodes beta-glucuronidase (Adbetagluc), delivered intravenously and into the brain parenchyma of MPS type VII mice, could provide long-term transgene expression and correction of lysosomal distension. We also tested whether systemic treatment with the immunosuppressive anti-CD40 ligand antibody, MR-1, affected transgene expression. We found substantial plasma beta-glucuronidase activity for over 9 weeks after gene transfer in the MR-1- treated group, with subsequent decline in activity corresponding to a delayed anti-beta-glucuronidase antibody response. At 16 weeks, near wild-type amounts of beta-glucuronidase activity and striking reduction of lysosomal pathology were detected in livers from mice that had received either MR-1 cotreatment or control antibody. In the lung and kidney, beta-glucuronidase activity was markedly higher for the MR-1-treated group. beta-Glucuronidase activity in the brain persisted independently of MR-1 treatment. Activity was intense in the injected hemisphere and was also evident in the noninjected cortex and striatum, with dramatic improvements in storage deposits in areas of both hemispheres. These results indicate that prolonged enzyme expression from transgenes delivered to deficient liver and brain can mediate pervasive correction and illustrate the potential for gene therapy of MPS and other lysosomal storage diseases.

Poly (lactic-glycolic) acid copolymer encapsulation of recombinant adenovirus reduces immunogenicity in vivo.

Adenovirus-mediated gene transfer has application to the treatment of diseases of the central nervous system. We demonstrate that a limitation to its use in vivo is an inability to redose to the brain. We show that one factor inhibiting re-dosing is the development of neutralizing anti-adenoviral antibodies. Encapsulation of recombinant adenovirus vectors in poly(lactic/glycolic acid) (PLGA) copolymer enables infection in vitro, in the presence of neutralizing antibodies and results in the release of viable virus for over 100 h. Importantly, encapsulated adenovirus also shows diminished immunogenicity in vivo. Mice immunized with encapsulated recombinant adenoviral vectors show a greater than 45-fold reduction in anti-adenovirus titers relative to non-encapsulated vectors. An extended release formulation of adenovirus that reduces viral immunogenicity and sequesters the viral particle form antibody exposure may improve in vivo efficacy.

Effects of macrophage depletion and anti-CD40 ligand on transgene expression and redosing with recombinant adenovirus.

The anti-CD40 ligand antibody MR-1, and macrophage-depleting liposomes were tested for their ability as transient immunosuppressive agents to: (1) prolong transgene expression; and (2) permit redosing after recombinant adenovirus infusion of mice. To test for effect on transgene duration, mice were infused with recombinant adenovirus coding for human factor IX (AdFIX), and plasma FIX levels monitored over time. Treatment with either agent significantly prolonged transgene expression. Persistence was accompanied by inhibition of anti-adenovirus (anti-Ad) IgG, and decreased IL-10 and IFN-gamma production from splenic lymphocytes re-exposed to virus particles in vitro. To test for effect on redosing, mice were given a primary infusion of recombinant adenovirus coding for bacterial beta-galactosidase (Ad beta gal), followed by secondary and tertiary infusions of AdFIX on days 24 and 63. Mice that had received MR-1 had low to undetectable anti-Ad on day 24, and efficient transduction occurred. Furthermore, FIX levels endured in these mice, with 40% retention of FIX on day 63, in contrast to rapid loss in naive controls. On day 63, the continuance of negligible anti-Ad levels correlated with successful tertiary transduction. These results suggest that both macrophage depletion and CD40 ligand blockade inhibit immune responses to recombinant adenovirus to slow decline of transgene expression, while only CD40 ligand blockade inhibits anti-Ad antibody generation sufficiently to allow redosing to the liver.

Involvement of nitric oxide in IFN-gamma-mediated reduction of microvessel smooth muscle cell proliferation.

Previous studies in our laboratory demonstrated that murine cerebral microvessel smooth muscle cells (SMC) activate syngeneic CD4+ T-cells in vitro. These T-cells, or their culture supernatants, in turn, strongly inhibit proliferation of the SMC. The present study focuses on IFN-gamma as a mediator of inhibition of SMC proliferation, and addresses the molecular mechanism of this inhibition. IFN-gamma profoundly reduced the proliferation of murine brain microvessel smooth muscle cells in vitro. Three lines of evidence indicate that nitric oxide contributed to this effect: (1) IFN-gamma-mediated inhibition of proliferation correlated with the quantity of nitrite, a stable breakdown product of nitric oxide, in culture supernatants; (2) the addition of N(g)- monomethyl-l-arginine, and inhibitor of nitric oxide synthesis, restored proliferation to control or near control levels; and (3) the addition of hemoglobin, which has a high affinity for, and thus sequesters nitric oxide, also resulted in significant restoration of the proliferative response. However, the nitric oxide donating chemical sodium nitro-prusside, at concentrations up to 100 microM, had no direct cytostatic effect. These results suggest that nitric oxide is a necessary but insufficient component in IFN-gamma-mediated inhibition of microvessel smooth muscle cell proliferation. TNF-alpha also stimulated nitric oxide production by the smooth muscle cells, but was not as potent as IFN-gamma at inhibiting proliferation. Knowledge of the physiological effects of lymphokines on cells of the brain microvasculature will contribute towards a better understanding of inflammatory processes in diseases such as multiple sclerosis and infectious encephalitis.

Rat splenocytes inhibit antigen-specific lymphocyte proliferation through a reactive nitrogen intermediate (RNI)-dependent mechanism and exhibit increased RNI production in response to IFN-gamma.

Rat splenocytes inhibited antigen-specific proliferation of primed lymph node cells in vitro. This inhibition resided in the plastic-adherent splenocyte fraction and was radioresistant, suggesting that the effect was due to macrophages. While this suppression was more evident if spleen cells were derived from immunized rats, spleen cells from normal rats were just as suppressive when added to cocultures at higher numbers. Proliferative responses were greatly enhanced in the presence of NG-monomethyl-L-arginine, a specific inhibitor of the nitric oxide synthetic pathway, and significant levels of nitrite (NO2-), a product of this pathway, were detected in culture supernatants in association with suppressed responses, supporting the notion that suppression was mediated by the L-arginine-dependent production of reactive nitrogen intermediates (RNI). When the splenocytes were physically separated from the responding lymph node cell population, high levels of NO2- were still detected but proliferative responses were no longer inhibited, suggesting that cell proximity or contact is necessary for delivery of the suppressive signal. Adherent splenocytes cultured alone produced low levels of NO2-. Addition of 1 to 50 U/ml IFN-gamma induced a dose-dependent increase in NO2- production, with the maximal level approximating that found in suppressed cocultures; TNF-alpha, IL-2, or LPS did not synergize with IFN-gamma to enhance NO2- production. These findings suggest that by activating macrophages to upregulate RNI synthesis, IFN-gamma-producing T cells may exert a negative influence over their own proliferation.

Myelin-liposome protection against experimental autoimmune encephalomyelitis is associated with reduced neuroantigen-specific T-cell-mediated responses.

Lewis rats undergo a relapsing paralytic disease upon challenge with spinal cord emulsified in complete Freund's adjuvant (CFA). Treatment with two intracardiac injections of liposomes composed of whole myelin significantly reduced the severity of disease. Protection was disease-specific since treatment with myelin liposomes did not protect Lewis rats against adjuvant arthritis (AA), a CNS-unrelated T-cell-mediated autoimmune disease. Myelin-liposome-treated, spinal cord/CFA-immunized rats displayed borderline reduction of delayed-type hypersensitivity (DTH) (ear swelling) reactions to myelin and myelin basic protein (MBP), but significantly reduced in vitro lymphnode cell proliferation in response to these antigens. Responses to purified protein derivative of Mycobacterium tuberculosis (PPD) were not reduced, emphasizing the antigen-specific nature of the myelin-liposome-mediated suppression. Spleen cell proliferative responses were inconsistent and often poor. However, when cultured in the presence of NG-monomethyl-L-arginine (MMA), antigen-specific proliferation of spleen cells from both treated and control rats was greatly enhanced, indicating that reactive nitrogen intermediates contributed to the decrease in spleen cell proliferation. Purified splenic T cells from treated rats displayed a pattern of proliferation similar to that of unseparated lymphnode cells. Treatment of rats with a single injection of myelin liposomes after recovery from the first clinical episode significantly reduced the severity of the relapses.

Validity and reliability of an ultrasound device for detecting carotid emboli.

Cardioembolic mechanisms cause 15 to 20% of all strokes and may account for the high incidence of neurological dysfunction associated with cardiopulmonary bypass. Accurate identification of high-risk subjects and/or surgical techniques would allow more effective testing and implementation of preventive or therapeutic measures to help reduce morbidity and mortality. This article reports on validity and reliability testing of a new emboli detection device that allows continuous monitoring of the common carotid artery. The instrument appears to be capable of detecting accurately particles of 193 mu or less in diameter and is highly reliable both within and between observers. In preliminary clinical use, the instrument also detected embolic signals in all patients monitored during cardiopulmonary bypass, while none were detected in healthy control subjects. These results establish the validity and reliability of a new emboli detection device and suggest its potential application to emboli detection monitoring during cardiopulmonary bypass.

Treatment of spinal cord-induced experimental allergic encephalomyelitis in the Lewis rat with liposomes presenting central nervous system antigens.

Chronic-relapsing experimental allergic encephalomyelitis (CR-EAE) in the Lewis rat, induced by the injection of spinal cord tissue in complete Freund's adjuvant (SC/CFA), was studied in vivo by treatment with liposomes containing central nervous tissue antigens, and in vitro by lymphocyte proliferation assays. Intracardiac administration of myelin basic protein (MBP) liposomes, galactocerebroside (GC) liposomes, or MBP + GC liposomes substantially reduced the clinical severity and/or delayed the onset of the initial phase of disease. Liposomes prepared from whole myelin provided even greater protection, and were effective at suppressing both the first disease episode and the relapses. These results indicate that while GC and MBP may play significant roles in the development of CR-EAE in the Lewis rat, immune responses to other antigens are probably also involved. Splenic and lymph node lymphocytes from MBP-GC liposome-treated rats, and splenic lymphocytes from cytochrome-GC (CYT-GC) liposome-treated rats, showed drastically reduced abilities to proliferate in response to MBP in culture. Spleen cells from both the MBP-GC- and CYT-GC-liposome-treated donors were able to actively suppress antigen-induced proliferation of MBP-primed lymphocytes. These findings suggest participation of both clonal anergy, and active suppressor cells in the liposome-mediated suppression of CR-EAE in the Lewis rat.