Retinal degeneration is slowed in transgenic rats by AAV-mediated delivery of FGF-2.

PURPOSE: We evaluated adeno-associated virus (AAV)-mediated gene transfer of basic fibroblast growth factor (FGF-2) as a therapy for photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa. METHODS: Recombinant adeno-associated virus vector (rAAV) incorporating a constitutive cytomegalovirus (CMV) promoter was used to transfer the bovine FGF-2 gene to photoreceptors. AAV was administered by subretinal injection to transgenic rats (TgN S334ter-4) at postnatal day 15 (P15). Control eyes were uninjected, injected with PBS, or AAV-LacZ. Eyes were examined by histopathology, morphometric analysis, and electroretinography at P60. RESULTS: Expression of recombinant FGF-2 slowed the rate of photoreceptor degeneration. Morphologic studies demonstrated significantly more photoreceptors surviving in eyes injected with AAV-FGF-2 than in controls. Insignificant rescue effects were seen in retinas injected with buffer only. No significant inflammatory response or neovascularization was detected. Electroretinographic (ERG) responses of eyes injected with AAV-FGF-2 were increased compared with uninjected eyes; however, these amplitudes were not significantly larger than eyes receiving an AAV-LacZ control vector. CONCLUSIONS: Transduction of retinal cells with AAV-FGF-2 reduces the rate of photoreceptor degeneration in an S334ter-4 animal model. Despite the lack of significantly increased ERG amplitudes from eyes expressing FGF-2, a greater number of surviving photoreceptors was demonstrated. Delivery of FGF-2 using recombinant AAV has potential as a therapy for retinal degeneration.

Dose response to a single intramuscular injection of recombinant adeno-associated virus-erythropoietin in monkeys.

BACKGROUND: Anemia is a significant problem in many disease states. Erythropoietin (Epo) has been used in the treatment of anemia associated with numerous chronic diseases. This study investigates the dose-response profiles of a single intramuscular (im) injection of a recombinant adeno-associated virus vector (rAAV) containing the Epo gene with the goal of achieving a sustained elevation of hematocrit (Hct). METHODS: Cynomolgus (cm) monkeys were given single injections of different doses of rAAV-cm-Epo. The biological effect of Epo gene expression was monitored by determining the Hct levels and circulating hormone levels by ELISA. Antibody to the rAAV capsid protein was also measured over the 41-week period of the experiment. RESULTS: Epo expression was noted only when 2 x 10(11) or more particles were injected. Epo was noted to be increased as soon as 1 week postinjection and was maximum in 6 to 8 weeks. This level of expression remained constant for nearly 20 weeks. Animals given the highest dose of rAAV developed a higher Hct over the first 8 weeks postinjection than those given an intermediate dose. However, the maximum levels of hemoglobin were the same. There was a weak correlation between amount of rAAV injected and capsid antibody response. CONCLUSIONS: AAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single im administration. Dose responses to rAAV-Epo are achievable, although a threshold inoculum of virus is necessary to produce an effect and the therapeutic window is narrow.

Adeno-associated virus-mediated delivery of erythropoietin leads to sustained elevation of hematocrit in nonhuman primates.

Recombinant adeno-associated virus encoding the monkey erythropoietin gene (rAAV-cm-Epo) was generated and tested for its potential to confer long-term expression of the gene product following intramuscular injection. A single intramuscular injection of 2 x 10(12) rAAV-cm-Epo particles into two baboons led to sustained high circulating Epo levels and a concomitant increase in hematocrit. The hematocrits reached 62 and 75% by week 10 (from pre-injection values of 38 and 40%, respectively) and remained elevated throughout the study period (28 weeks). Circulating Epo levels were also elevated throughout the study period. Our data demonstrate the potential for long-term gene expression in large animals by a single intramuscular injection of a recombinant adeno-associated virus (rAAV) vector.

Lipitoids--novel cationic lipids for cellular delivery of plasmid DNA in vitro.

BACKGROUND: Although synthetic nonviral vectors hold promise for the delivery of plasmid DNA, their gene-transfer efficiencies are far from matching those of viruses. To systematically investigate the structure-activity relationship of cationic lipids, a small library of cationic lipid-peptoid conjugates (lipitoids) was synthesized. The compounds were evaluated for their ability to form complexes with plasmid DNA and to mediate DNA transfer in vitro. RESULTS: Lipid-peptoid conjugates were conveniently prepared in high yield using solid-phase synthesis. Several lipitoids condensed plasmid DNA into 100 nm spherical particles and protected the DNA and DNase digestion. A subset of lipitoids with a repeated (aminoethyl, neutral, neutral) sidechain trimer motif conjugated with dimyristoyl phosphatidyl-ethanolamine (DMPE) mediated DNA transfer with high efficiency. CONCLUSIONS: Automated solid-phase synthesis of cationic lipids allowed the rapid synthesis of a diverse set of transfection reagents. The most active compound DMPE-(Nae-Nmpe-Nmpe)3 (Nae, N-aminoethyl glycine; Nmpe, N-p-methoxyphenethyl-glycine) is more efficient than lipofectin or DMRIE-C (two commercial cationic lipid transfection reagents) and is active in the presence and absence of serum. The activity in the presence of serum suggests potential for applications in vivo.

Transient immunosuppression allows transgene expression following readministration of adeno-associated viral vectors.

Adeno-associated viral (AAV) vectors have much promise in gene therapy. Among the many properties that make AAV an ideal vector for gene therapy are its ability to infect both dividing and nondividing cells and the longevity of expression in tissues such as brain, skeletal muscle, and liver. However, like other viral vectors, readministration of vector is limited because of the host's immune response to viral components of the vector. Using class I, class II, and CD40 ligand (CD40L)-deficient mice, we demonstrate that neutralizing antibodies to the viral capsid proteins prevent transgene expression following readministration of rAAV vectors. Transient immunosuppression of mice by treatment with antibody to CD4 at the time of primary infection allowed transgene expression after readministration of rAAV vectors to animals. Transient immunosuppression with antibody to CD40L had only a modest effect on the efficacy of readministration. The ability to readminister virus was inversely correlated with both AAV capsid enzyme-linked immunosorbent assay titers and AAV neutralizing antibody titers. These studies demonstrate that readministration of rAAV can be accomplished by down regulating the anti-AAV immune response and suggest the use of repeated administration of rAAV as a viable form of therapy for the treatment of chronic diseases.

Genetic immunization with adeno-associated virus vectors expressing herpes simplex virus type 2 glycoproteins B and D.

Intramuscular injection of mice with an adeno-associated virus (AAV) vector expressing herpes simplex virus type 2 glycoprotein B led to the generation of both gB-specific major histocompatibility complex class I-restricted cytotoxic T lymphocytes and anti-gB antibody. AAV-mediated immunization was more potent than plasmid DNA or protein in generating antibody responses.

Long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin.

The ob/ob mouse is genetically deficient in leptin and exhibits a phenotype that includes obesity and non-insulin-dependent diabetes mellitus. This phenotype closely resembles the morbid obesity seen in humans. In this study, we demonstrate that a single intramuscular injection of a recombinant adeno-associated virus (AAV) vector encoding mouse leptin (rAAV-leptin) in ob/ob mice leads to prevention of obesity and diabetes. The treated animals show normalization of metabolic abnormalities including hyperglycemia, insulin resistance, impaired glucose tolerance, and lethargy. The effects of a single injection have lasted through the 6-month course of the study. At all time points measured the circulating levels of leptin in the serum were similar to age-matched control C57 mice. These results demonstrate that maintenance of normal levels of leptin (2-5 ng/ml) in the circulation can prevent both the onset of obesity and associated non-insulin-dependent diabetes. Thus a single injection of a rAAV vector expressing a therapeutic gene can lead to complete and long-term correction of a genetic disorder. Our study demonstrates the long-term correction of a disease caused by a genetic defect and proves the feasibility of using rAAV-based vectors for the treatment of chronic disorders like obesity.

Angiotensin II type 1 receptor signals through Raf-1 by a protein kinase C-dependent, Ras-independent mechanism.

To understand the molecular mechanism by which the angiotensin II (AII) type 1 receptor (AT1 receptor) transduces its biological signal, we examined the role of various signaling molecules involved in AT1 receptor signaling in Chinese hamster ovary cells stably transfected with the AT1 receptor. AT1 receptor-transfected cells responded to AII treatment by inhibiting adenylyl cyclase, increasing the intracellular Ca2+ concentration, and activating protein kinase C (PKC) alpha and PKC epsilon. AII also activated the c-fos gene and mitogen-activated protein (MAP) kinases. The activation of PKC, the c-fos gene, and MAP kinases was blocked by inhibition of PKC induced by pretreatment with 12-O-tetradecanoylphorbol-13-acetate but not by pretreatment with pertussis toxin, suggesting that PKC couples to the activation of the the c-fos gene and MAP kinases. In addition, AII activated Raf-1 and MAP kinase kinase in a PKC-dependent manner. A dominant negative mutant of Ras had no effect on AII-induced MAP kinase or c-fos gene activation. Thus, the AT1 receptor signals through Raf-1 and its downstream signaling molecules by a PKC-dependent mechanism that does not involve Ras activation.

Viability of transgenic mice expressing a platelet derived growth factor (PDGF) antagonist in plasma.

BACKGROUND: Many studies have implicated PDGF in the development of diseases such as atherosclerosis. Previously, we showed in tissue culture that the soluble extracellular domain of the PDGF beta-receptor is capable of binding BB-PDGF with high affinity; therefore antagonizing the ability of BB-PDGF to stimulate cell growth. METHODS: This work describes the efforts of expressing the soluble extracellular domain of the PDGF beta-receptor in transgenic mice. Driven by the albumin promoter, which is activated relatively late during embryonic development, the secreted form of the PDGF receptor protein was detected in plasma of the homozygous mice at a high concentration (approximately 60 micrograms/microL or approximately 545 nm). RESULTS: Plasma from these transgenic mice was capable of blocking PDGF-induced receptor autophosphorylation in tissue culture. The mice appeared to be healthy, demonstrating that full PDGF beta-receptor function is not required for viability. CONCLUSION: By expressing a high level of a soluble form of the extracellular domain of the PDGF receptor in transgenic mice, we have established a novel animal model that will allow us to gain insight into the role of the PDGF receptor in vascular diseases and other diseases involving PDGF stimulated cell proliferation.

Receptor tyrosine kinase stimulates cell-matrix adhesion by phosphatidylinositol 3 kinase and phospholipase C-gamma 1 pathways.

Receptor tyrosine kinases are known to be important in growth and differentiation. We have recently found that c-kit, the tyrosine kinase receptor for steel factor, also regulates cell-matrix adhesion. Because Steel factor helps regulate cell migration and localization, this may be an important biologic function. Integrin adhesiveness is regulated within minutes by c-kit. The signaling pathways for tyrosine kinase stimulation of integrin adhesiveness and their relation to pathways that regulate growth and differentiation over much longer time periods remain uncharacterized. We have studied the effector pathways by which receptor tyrosine kinases regulate cell-matrix adhesion using wild-type and mutant forms of the platelet-derived growth factor (PDGF) receptor, which is closely related to c-kit. The PDGF receptor expressed in mast cells is as potent as c-kit in stimulating adhesion to fibronectin. We show that induction of adhesion is regulated through two independent pathways of phosphatidylinositol 3 kinase (PI3K) and phospholipase C-gamma 1 (PLC gamma)-protein kinase C by elimination of autophosphorylation sites required for activation of PI3K and PLC gamma or in combination with downregulation of protein kinase C or wortmannin. By contrast, a receptor mutated in both the PI3K and PLC gamma association sites can still stimulate mast cell growth, indicating a crucial role of these effector molecules in regulating adhesion rather than cell growth.

Identification of a domain in the angiotensin II type 1 receptor determining Gq coupling by the use of receptor chimeras.

The angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors belong to the seven transmembrane receptor superfamily. Previous studies have suggested that the AT1R couples to a Gq signaling pathway, whereas the AT2R does not associate with Gq. To identify the role that individual intracellular domains play in AT1R function, AT1R/AT2R chimeric receptors were prepared by substitution of intracellular loops. CHO cells expressing these chimeras were used to test angiotensin II-induced c-fos expression and Ca2+ mobilization which are involved in the AT1R signaling pathway through Gq coupling. Substitution of the second intracellular loop (IC2) and the cytoplasmic tail between the two receptors did not affect AT1R function. However, exchange of the third intracellular loop (IC3) resulted in the loss of function in the AT1R and conferred to the AT2R the ability to constitutively activate the fos promoter. These findings suggest that the third intracellular loop of the AT1R is critical for Gq coupling. Substitution of discrete amino acid sequences of the third intracellular loop indicate that its N-terminal and C-terminal portions, especially the seven amino acids 219-225 in the N-terminal portion, are important for AT1R function, and that the intermediate portion of this loop is not required for Gq coupling.

Identification of Tyr-397 as the primary site of tyrosine phosphorylation and pp60src association in the focal adhesion kinase, pp125FAK.

A number of cellular processes, such as proliferation, differentiation, and transformation, are regulated by cell-extracellular matrix interactions. Previous studies have identified a novel tyrosine kinase, the focal adhesion kinase p125FAK, as a component of cell adhesion plaques. p125FAK was identified as a 125-kDa tyrosine-phosphorylated protein in cells transformed by the v-src oncogene. p125FAK is an intracellular protein composed of three domains: a central domain with homology to protein tyrosine kinases, flanked by two noncatalytic domains of 400 amino acids which bear no significant homology to previously cloned proteins. p125FAK is believed to play an important regulatory role in cell adhesion because it localizes to cell adhesion plaques and because its phosphorylation on tyrosine residues is regulated by binding of cell surface integrins to the extracellular matrix. Recent studies have shown that Src, through its SH2 domain, stably associates with pp125FAK and that this association prevents dephosphorylation of pp125FAK in vitro by protein tyrosine phosphatases. In this report, we identify Tyr-397 as the primary in vivo and in vitro site of p125FAK tyrosine phosphorylation and association with Src. Substituting phenylalanine for tyrosine at position 397 significantly reduces p125FAK tyrosine phosphorylation and association with Src but does not abolish p125FAK kinase activity. In addition, p125FAK kinase is able to trans-phosphorylate Tyr-397 in vitro in a kinase-deficient p125FAK variant. Phosphorylation of Tyr-397 provides a site [Y(P)AEI] that fits the consensus sequence for the binding of Src.

Platelet-derived growth factor triggers translocation of the insulin-regulatable glucose transporter (type 4) predominantly through phosphatidylinositol 3-kinase binding sites on the receptor.

Insulin is the only known hormone which rapidly stimulates glucose uptake in target tissues, mainly by translocation to the cell surface of the intracellular insulin-regulatable glucose transporter (glucose transporter type 4, GLUT4). We have developed a cell line for direct, sensitive detection of GLUT4 on the cell surface. We have suggested that insulin-activated phosphatidylinositol (PI) 3-kinase may be involved in the signaling pathway of insulin-stimulated GLUT4 translocation. We report that platelet-derived growth factor (PDGF), which stimulates PI 3-kinase activity, triggers GLUT4 translocation in Chinese hamster ovary (CHO) cells stably overexpressing the PDGF receptor and in 3T3-L1 mouse adipocytes. Using mutant PDGF receptors that cannot bind to Ras-GTPase-activating protein, phospholipase C-gamma, and PI 3-kinase, respectively, we obtained evidence that PI 3-kinase binding sites play a key role in the signaling pathway of PDGF-stimulated GLUT4 translocation in the CHO cell system.

Activation of Na+/H+ exchange by platelet-derived growth factor involves phosphatidylinositol 3'-kinase and phospholipase C gamma.

The effect of site-specific mutations in the mouse platelet-derived growth factor (PDGF) beta-receptor on activation of the Na+/H+ exchanger was examined in normal murine mammary gland epithelial (NMuMG) and Chinese hamster ovary (CHO) cells. These cells, which do not normally express PDGF receptors, were stably transfected with PDGF beta-receptor cDNA. Intracellular pH and Ca2+ were monitored using fluorescent probes. In both NMuMG and CHO cells expressing wild-type PDGF beta-receptors, PDGF B/B activated the amiloride-sensitive Na+/H+ exchanger. In both cell types, cell alkalinization was reduced by approximately 50% with a receptor mutant Y708F,Y719F which cannot bind phosphatidylinositol (PI) 3'-kinase. An inhibitor of PI 3'-kinase, LY294002, also inhibited alkalinization by 43% in cells with wild-type, but not Y708F,Y719F receptors. PDGF-induced intracellular Ca2+ release was not affected by this mutation. Both alkalinization and Ca2+ release were reduced by nearly 100% with the mutant Y977F,Y989F, which cannot bind phospholipase C gamma (PLC gamma). Y739F, a mutant that fails to bind the GTPase-activating protein did not affect PDGF-induced alkalinization. In protein kinase C (PKC) down-regulated NMuMG cells (wild-type receptor), PDGF no longer activated the Na+/H+ exchanger. In contrast, in PKC down-regulated CHO cells (wild-type receptor), PDGF-induced alkalinization was attenuated by only 37%. This residual activity was unaffected by the Y708F,Y719F mutation, but was completely eliminated by removal of medium Ca2+. These findings indicate that phospholipase C gamma is essential for activation of Na+/H+ exchange. PI 3'-kinase participates in PKC-dependent activation of Na+/H+ exchange by PDGF. In CHO cells, there is a second, Ca(2+)-dependent mechanism for activation of the exchanger.

The interaction of small domains between the subunits of phosphatidylinositol 3-kinase determines enzyme activity.

Previous studies have suggested that the two subunits of phosphatidylinositol (PI) 3-kinase, p85 and p110, function as localizing and catalytic subunits, respectively. Using recombinant p85 and p110 molecules, we have reconstituted the specific interaction between the two subunits of mouse PI 3-kinase in cells and in vitro. We have previously shown that the region between the two Src homology 2 (SH2) domains of p85 is able to form a functional complex with the 110-kDa subunit in vivo. In this report, we identify the corresponding domain in p110 which directs the binding to p85. We demonstrate that the interactive domains in p85 and p110 are less than 103 and 124 amino acids, respectively, in size. We also show that the association of p85 and p110 mediated by these domains is critical for PI 3-kinase activity. Surprisingly, a complex between a 102-amino-acid segment of p85 and the full-length p110 molecule is catalytically active, whereas p110 alone has no activity. In addition to the catalytic domain in the carboxy-terminal region, 123 amino acids at the amino terminus of p110 were required for catalytic activity and were sufficient for the interaction with p85. These results indicate that the 85-kDa subunit, previously thought to have only a linking role in localizing the p110 catalytic subunit, is an important component of the catalytic complex.

Evidence for both tyrosine kinase and G-protein-coupled pathways leading to starfish egg activation.

To investigate possible pathways leading to egg activation at fertilization, the ability of exogenously introduced tyrosine kinase and G-protein-coupled receptors to mimic events of fertilization was examined. Oocytes of the starfish Asterina miniata were injected with RNA for a chimeric receptor consisting of the extracellular domain of the beta form of the mouse platelet-derived growth factor (PDGF) receptor and the transmembrane/intracellular domain of the human fibroblast growth factor (FGF) receptor, or with RNA for the rat serotonin 1c receptor. These oocytes were cultured for 1 to 3 days and then matured with 1-methyladenine. In response to PDGF or serotonin, the injected eggs underwent responses like those at fertilization: cortical granule exocytosis, a rise in intracellular free calcium, and DNA synthesis. Some of these artificially activated eggs cleaved, and some of the PDGF-activated eggs were observed to form larvae. A PDGF/FGF receptor with a point mutation which eliminated its ability to interact with phospholipase C-gamma did not cause fertilization-like responses. Thus components of a signaling pathway involving phospholipase C-gamma, characteristic of tyrosine kinase receptors, as well as components of a pathway involving a G-protein and phospholipase C-beta, characteristic of G-protein-coupled receptors, appear to be present in starfish eggs. Either or both could function in egg activation at fertilization.

Regulation of chemotaxis by the platelet-derived growth factor receptor-beta.

Chemotaxis is an important component of wound healing, development, immunity and metastasis, yet the signalling pathways that mediate chemotaxis are poorly understood. Platelet-derived growth factor (PDGF) acts both as a mitogen and a chemoattractant. Upon stimulation, the tyrosine kinase PDGF receptor-beta (PDGFR-beta) autophosphorylates and forms a complex that includes SII2(Src homology 2)-domain-containing proteins such as the phosphatidylinositol-specific phospholipase C-gamma, Ras-GTPase-activating protein (GAP), and phosphatidylinositol-3-OH kinase. Specific tyrosine-to-phenylalanine substitutions in the PDGFR-beta can prevent binding of one SH2-domain-containing protein without affecting binding of other receptor-associated proteins. Here we use phospholipase C-gamma and PDGFR-beta mutants to map specific tyrosines involved in both positive and negative regulation of chemotaxis towards the PDGF-BB homodimer. Our results indicate that a delicate balance of migration-promoting (phospholipase C-gamma and phosphatidylinositol-3-OH kinase) and migration-suppressing (GAP) activities are recruited by the PDGFR-beta to drive chemotaxis towards PDGF-BB.

Dominant-negative mutations of platelet-derived growth factor (PDGF) receptors. Inhibition of receptor function by ligand-dependent formation of heterodimers between PDGF alpha- and beta-receptors.

We showed previously that a truncated form of the platelet-derived growth factor (PDGF) beta-receptor lacking its kinase region can form a nonfunctional heterodimer with the wild-type beta-receptor and thereby inhibit its signal transduction. In this paper we investigated whether the truncated form of either alpha- or beta-receptor could block the function of the other type of wild-type PDGF receptor. When the truncated alpha-receptor was expressed in Xenopus oocytes in excess over either the wild-type alpha- or beta-receptor, the Ca2+ mobilization signal elicited by either the wild-type alpha- or beta-receptor was completely blocked. The truncated beta-receptor abolished signaling by the wild-type alpha-receptor in response to PDGF-AB or -BB. However signal transduction by the alpha-receptor in response to PDGF-AA was not affected by the truncated beta-receptor. In the presence of PDGF-AB or -BB, both the wild-type and truncated beta-receptors formed a heterologous complex with the alpha-receptor in intact cells. A kinase-inactive beta-receptor (an ATP-binding site mutation) became cross-phosphorylated on tyrosine residues by the co-expressed wild-type alpha-receptor in response to PDGF-AB or -BB but not in response to PDGF-AA. These findings indicate that the alpha-and beta-receptors interact in response to PDGF-AB or -BB and are consistent with the hypothesis that the truncated alpha-receptor inhibits function of the wild-type beta-receptor through formation of a ligand-dependent nonfunctional alpha beta-heterodimer. A similar heterodimer can form between the truncated beta-receptor and the wild-type alpha-receptor. These observations provide useful information for future studies using dominant-negative mutations of PDGF receptors to selectively inhibit the actions of specific PDGFs in animals.

Signal transduction through a biomolecular receptor tyrosine protein kinase composed of a platelet-derived growth factor receptor-CD4 chimera and the nonreceptor tyrosine protein kinase Lck.

We have generated a novel "receptor tyrosine kinase" by fusing the extracellular and transmembrane domain of the mouse platelet-derived growth factor receptor (PDGFR) to the cytoplasmic domain of CD4 and coexpressing the construct with the murine cytoplasmic tyrosine protein kinase p56lck. NMuMG cells, which are mouse mammary gland epithelial cells that lack endogenous platelet-derived growth factor (PDGF) receptor expression, were stably transfected with both PDGFR-CD4 and p56lck. The PDGFR-CD4 chimeric protein was expressed at the cell surface and formed a complex with p56lck. Addition of PDGF to these cells led to increased tyrosine phosphorylation of a 56-kDa protein likely to be p56lck and several unidentified cellular proteins. The enzymatic activity of p56lck was increased after treatment with PDGF, indicating that dimerization (or oligomerization) mediated by ligand binding at the cell surface is capable of inducing the activation not only of receptor tyrosine kinases but nonreceptor tyrosine kinases as well. However, the PDGFR-CD4.p56lck complex was, in contrast to the wild type PDGF receptor, not able to induce a PDGF-dependent mitogenic response or DNA synthesis in NMuMG cells. Analysis of several known substrates of the PDGFR-signaling pathway indicates an early block in the transduction of the signal generated by p56lck.