RGD capsid modification enhances mucosal protective immunity of a non-human primate adenovirus vector expressing Pseudomonas aeruginosa OprF.

Replication-deficient adenoviral (Ad) vectors of non-human serotypes can serve as Ad vaccine platforms to circumvent pre-existing anti-human Ad immunity. We found previously that, in addition to that feature, a non-human primate-based AdC7 vector expressing outer membrane protein F of P. aeruginosa (AdC7OprF) was more potent in inducing lung mucosal and protective immunity compared to a human Ad5-based vector. In this study we analysed if genetic modification of the AdC7 fibre to display an integrin-binding arginine-glycine-aspartic acid (RGD) sequence can further enhance lung mucosal immunogenicity of AdC7OprF. Intratracheal immunization of mice with either AdC7OprF.RGD or AdC7OprF induced robust serum levels of anti-OprF immunoglobulin (Ig)G up to 12 weeks that were higher compared to immunization with the human vectors Ad5OprF or Ad5OprF.RGD. OprF-specific cellular responses in lung T cells isolated from mice immunized with AdC7OprF.RGD and AdC7OprF were similar for T helper type 1 (Th1) [interferon (IFN)-γ in CD8(+) and interleukin (IL)-12 in CD4(+)], Th2 (IL-4, IL-5 and IL-13 in CD4(+)) and Th17 (IL-17 in CD4(+)). Interestingly, AdC7OprF.RGD induced more robust protective immunity against pulmonary infection with P. aeruginosa compared to AdC7OprF or the control Ad5 vectors. The enhanced protective immunity induced by AdC7OprF.RGD was maintained in the absence of alveolar macrophages (AM) or CD1d natural killer T cells. Together, the data suggest that addition of RGD to the fibre of an AdC7-based vaccine is useful to enhance its mucosal protective immunogenicity.

Assessment of disease severity in late infantile neuronal ceroid lipofuscinosis using multiparametric MR imaging.

BACKGROUND AND PURPOSE: LINCL is a uniformly fatal lysosomal storage disease resulting from mutations in the CLN2 gene that encodes for tripeptidyl peptidase 1, a lysosomal enzyme necessary for the degradation of products of cellular metabolism. With the goal of developing quantitative noninvasive imaging biomarkers sensitive to disease progression, we evaluated a 5-component MR imaging metric and tested its correlation with a clinically derived disease-severity score. MATERIALS AND METHODS: MR imaging parameters were measured across the brain, including quantitative measures of the ADC, FA, nuclear spin-spin relaxation times (T2), volume percentage of CSF (%CSF), and NAA/Cr ratios. Thirty MR imaging datasets were prospectively acquired from 23 subjects with LINCL (2.5-8.4 years of age; 8 male/15 female). Whole-brain histograms were created, and the mode and mean values of the histograms were used to characterize disease severity. RESULTS: Correlation of single MR imaging parameters against the clinical disease-severity scale yielded linear regressions with R2 ranging from 0.25 to 0.70. Combinations of the 5 biomarkers were evaluated by using PCA. The best combination included ADC, %CSF, and NAA/Cr (R2=0.76, P<.001). CONCLUSIONS: The multiparametric disease-severity score obtained from the combination of ADC, %CSF, and NAA/Cr whole-brain MR imaging techniques provided a robust measure of disease severity, which may be useful in clinical therapeutic trials of LINCL in which an objective assessment of therapeutic response is desired.

Glutathione S-transferase copy number variation alters lung gene expression.

The glutathione S-transferase (GST) enzymes catalyse the conjugation of xenobiotics to glutathione. Based on reports that inherited copy number variations (CNVs) modulate some GST gene expression levels, and that the small airway epithelium (SAE) and alveolar macrophages (AMs) are involved early in the pathogenesis of smoking-induced lung disease, we asked: do germline CNVs modulate GST expression levels in SAE and AMs? Microarrays were used to survey GST gene expression and determine CNVs genotypes in SAE and AMs obtained by bronchoscopy from current smokers and nonsmokers. 26% of subjects were null for both GSTM1 alleles, with reduced GSTM1 mRNA levels seen in both SAE and AMs. 30% of subjects had homozygous deletions of GSTT1, with reduced mRNA levels in both tissues. Interestingly, GSTT2B exhibited homozygous deletion in the blood of 27% of subjects and was not expressed in SAE in the remainder of subjects, but was expressed in AMs of heterozygotes and wild-type subjects, proportionate to genotype. These data show a germline CNV-mediated linear relationship of genotype with expression level, suggesting minimal compensation of gene expression levels in heterozygotes, consistent with GST polymorphisms playing a role in the risk of smoking-associated, xenobiotic-induced lung disease.

Persistent expression of biologically active anti-HER2 antibody by AAVrh.10-mediated gene transfer.

Trastuzumab (Herceptin) is a recombinant humanized monoclonal antibody (mAb) directed against an extracellular region of the human epidermal growth-factor receptor type 2 (HER2) protein. We hypothesized that a single adeno-associated virus (AAV)-mediated genetic delivery of an anti-HER2 antibody should be effective in mediating long-term production of anti-HER2 and in suppressing the growth of human tumors in a xenograft model in nude mice. The adeno-associated virus gene transfer vector AAVrh.10alphaHER2 was constructed based on a non-human primate AAV serotype rh.10 to express the complementary DNAs for the heavy and light chains of mAb 4D5, the murine precursor to trastuzumab. The data show that genetically transferred anti-HER2 selectively bound human HER2 protein and suppressed the proliferation of HER2(+) tumor cell lines. A single administration of AAVrh.10alphaHER2 provided long-term therapeutic levels of anti-HER2 antibody expression without inducing an anti-idiotype response, suppressed the growth of HER2(+) tumors and increased the survival of tumor bearing mice. In the context that trastuzumab therapy requires frequent and repeated administration, this strategy might be developed as an alternate platform for delivery of anti-HER2 therapy.

Assessing disease severity in late infantile neuronal ceroid lipofuscinosis using quantitative MR diffusion-weighted imaging.

BACKGROUND AND PURPOSE: Late infantile neuronal ceroid lipofuscinosis (LINCL), a form of Batten disease, is a fatal neurodegenerative genetic disorder, diagnosed via DNA testing, that affects approximately 200 children in the United States at any one time. This study was conducted to evaluate whether quantitative data derived by diffusion-weighted MR imaging (DWI) techniques can supplement clinical disability scale information to provide a quantitative estimate of neurodegeneration, as well as disease progression and severity. MATERIALS AND METHODS: This study prospectively analyzed 32 DWI examinations from 18 patients having confirmed LINCL at various stages of disease. A whole-brain apparent diffusion coefficient (ADC) histogram was fitted with a dual Gaussian function combined with a function designed to model voxels containing a partial volume fraction of brain parenchyma versus CSF. Previously published whole-brain ADC values of age-matched control subjects were compared with those of the LINCL patients. Correlations were tested between the peak ADC of the fitted histogram and patient age, disease severity, and a CNS disability scale adapted for LINCL. RESULTS: ADC values assigned to brain parenchyma were higher than published ADC values for age-matched control subjects. ADC values between patients and control subjects began to differ at 5 years of age based on 95% confidence intervals. ADC values had a nearly equal correlation with patient age (R2=0.71) and disease duration (R2=0.68), whereas the correlation with the central nervous system disability scale (R2=0.27) was much weaker. CONCLUSION: This study indicates that brain ADC values acquired using DWI may be used as an independent measure of disease severity and duration in LINCL.

Neurological deterioration in late infantile neuronal ceroid lipofuscinosis.

BACKGROUND: Late infantile neuronal ceroid lipofuscinosis (LINCL) is associated with progressive degeneration of the brain and retina starting in early childhood. METHODS: Thirty-two individual neurologic, ophthalmologic, and CNS imaging (MRI and MRS) assessments of 18 children with LINCL were analyzed. Disease severity was followed by two rating scales, one previously established but modified to solely assess the brain and exclude the retinal disease (modified Hamburg LINCL scale), and a newly developed scale, with expanded evaluation of the CNS impairment (Weill Cornell LINCL scale). RESULTS: For the 18 children, the Weill Cornell scale yielded a closer correlation with both age and time since initial clinical manifestation of the disease than did the modified Hamburg scale. There were no significant differences as a function of age or time since initial manifestation of the disease in the rating scales among the most frequent CLN2 mutations (G3556C, 56% of all alleles or C3670T, 22% of all alleles). Measurements of cortical MRS N-acetyl-aspartate content, MRI ventricular, gray matter and white matter volume, and cortical apparent diffusion coefficient correlated to a variable degree with the age of the children and the time since initial clinical manifestation of the disease. All imaging measurements correlated better with the Weill Cornell CNS scale compared to the modified Hamburg LINCL scale. CONCLUSION: The data suggest that the Weill Cornell late infantile neuronal ceroid lipofuscinosis (LINCL) scale, together with several of the MRI measurements, may be useful in the assessment of severity and progression of LINCL and for the evaluation of novel therapeutic strategies.

AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in long-term TPP-I expression compatible with therapy for LINCL.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal, autosomal recessive disease resulting from mutations in the CLN2 gene with consequent deficiency in its product tripeptidyl peptidase I (TPP-I). In the central nervous system (CNS), the deficiency of TPP-I results in the accumulation of proteins in lysosomes leading to a loss of neurons causing progressive neurological decline, and death by ages 10-12 years. To establish the feasibility of treating the CNS manifestations of LINCL by gene transfer, an adeno-associated virus 2 (AAV2) vector encoding the human CLN2 cDNA (AAV2CUhCLN2) was assessed for its ability to establish therapeutic levels of TPP-I in the brain. In vitro studies demonstrated that AAV2CUhCLN2 expressed CLN2 and produced biologically active TPP-I protein of which a fraction was secreted as the pro-TPP-I precursor and was taken up by nontransduced cells (ie, cross-correction). Following AAV2-mediated CLN2 delivery to the rat striatum, enzymatically active TPP-I protein was detected. By immunohistochemistry TPP-I protein was detected in striatal neurons (encompassing nearly half of the target structure) for up to 18 months. At the longer time points following striatal administration, TPP-I-positive cell bodies were also observed in the substantia nigra, frontal cerebral cortex and thalamus of the injected hemisphere, and the frontal cerebral cortex of the noninjected hemisphere. These areas of the brain contain neurons that extend axons into the striatum, suggesting that CNS circuitry may aid the distribution of the gene product. To assess the feasibility of human CNS delivery, a total of 3.6 x 10(11) particle units of AAV2CUhCLN2 was administered to the CNS of African green monkeys in 12 distributed doses. Assessment at 5 and 13 weeks demonstrated widespread detection of TPP-I in neurons, but not glial cells, at all regions of injection. The distribution of TPP-I-positive cells was similar between the two time points at all injection sites. Together, these data support the development of direct CNS gene transfer using an AAV2 vector expressing the CLN2 cDNA for the CNS manifestations of LINCL.

Intratumoral administration of low doses of an adenovirus vector encoding tumor necrosis factor alpha together with naive dendritic cells elicits significant suppression of tumor growth without toxicity.

Although tumor necrosis factor alpha (TNF-alpha) is a potent cytokine with a myriad of innate immune antitumor properties, systemic administration of TNF-alpha is associated with significant toxicity, limiting the use of the TNF-alpha protein as an antitumor therapeutic. On the basis of the knowledge that dendritic cells (DCs) play a central role in initiating antitumor adaptive immune responses, we hypothesized that intratumoral administration of low doses of an adenovirus encoding TNF-alpha (AdTNF-alpha) together with syngeneic DCs would act synergistically to suppress preexisting tumors. As a model, four different tumor cell lines, all resistant in vitro to the TNF-alpha protein, were implanted in syngeneic mice, and established tumors received intratumor AdTNF-alpha alone or in combination with DCs. At high doses (10(9) PFU), AdTNF-alpha alone suppressed tumor growth, but was associated with systemic toxicity. A 100-fold lower AdTNF-alpha concentration (10(7) PFU) or high doses of the control vector AdNull had no systemic toxicity, but also minimal suppression of tumor growth. In contrast, local administration of the low dose (10(7) PFU) of AdTNF-alpha in combination with syngeneic DCs (AdTNF-alpha + DCs) elicited marked tumor suppression without toxicity. Administration of AdTNF-alpha + DCs into tumors elicited tumor-specific cytotoxic T cells and protected animals against subsequent challenge with the same tumor, suggesting that AdTNF-alpha + DC therapy induced tumor-specific adaptive immune host responses. Consistent with this concept, studies with syngeneic knockout mice showed that MHC class I molecules on DCs as well as CD8(+) T cells were necessary for the antitumor effect of intratumor AdTNF-alpha + DCs. These data demonstrate that the combination of intratumoral administration of the TNF-alpha cDNA together with naive DCs can evoke tumor suppression without systemic toxicity, providing a new paradigm for the use of TNF-alpha as antitumor therapy.

Feasibility of gene therapy for late neuronal ceroid lipofuscinosis.

Late infantile neuronal ceroid lipofuscinosis is a progressive childhood neurodegenerative disorder characterized by intracellular accumulation of autofluorescent material resembling lipofuscin in neuronal cells. This report summarizes the new therapies under consideration for late infantile neuronal ceroid lipofuscinosis, with a focus on strategies for in vivo gene therapy for the retinal and central nervous system manifestations of the disease.

Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth.

The role of bone marrow (BM)-derived precursor cells in tumor angiogenesis is not known. We demonstrate here that tumor angiogenesis is associated with recruitment of hematopoietic and circulating endothelial precursor cells (CEPs). We used the angiogenic defective, tumor resistant Id-mutant mice to show that transplantation of wild-type BM or vascular endothelial growth factor (VEGF)-mobilized stem cells restore tumor angiogenesis and growth. We detected donor-derived CEPs throughout the neovessels of tumors and Matrigel-plugs in an Id1+/-Id3-/- host, which were associated with VEGF-receptor-1-positive (VEGFR1+) myeloid cells. The angiogenic defect in Id-mutant mice was due to impaired VEGF-driven mobilization of VEGFR2+ CEPs and impaired proliferation and incorporation of VEGFR1+ cells. Although targeting of either VEGFR1 or VEGFR2 alone partially blocks the growth of tumors, inhibition of both VEGFR1 and VEGFR2 was necessary to completely ablate tumor growth. These data demonstrate that recruitment of VEGF-responsive BM-derived precursors is necessary and sufficient for tumor angiogenesis and suggest new clinical strategies to block tumor growth.

Cross-strain protection against clinical and laboratory strains of Pseudomonas aeruginosa mediated by dendritic cells genetically modified to express CD40 ligand and pulsed with specific strains of Pseudomonas aeruginosa.

We have shown that dendritic cells (DCs) genetically engineered with a recombinant adenovirus vector (Ad) to express CD40 ligand (CD40L) elicit specific humoral immunity against the Pseudomonas aeruginosa laboratory strain PAO1, without CD4(+) T cell help. In the present study, using several different strains of P. aeruginosa, we examine whether this strategy is generally applicable to enhancing clinically relevant pathogen-specific immunity. Mice immunized with DCs modified with CD40L and pulsed with heat-killed P. aeruginosa clinical strain PA514, originally isolated from the sputum of an individual with cystic fibrosis, survived lethal respiratory challenge with PA514-impregnated agar beads. Consistent with this effective in vivo protection, the immunized mice generated high levels of serum isotype-switched antibodies directed against PA514 without concomitant nonspecific elevations of total serum immunoglobulin levels. The CD40L genetically engineered DCs pulsed with seven of eight different strains of P. aeruginosa afforded significant, albeit variable, cross-protection against lethal respiratory challenge with a clinical (PA514) or laboratory (PAO1) strain of P. aeruginosa. CD40L genetically modified DCs pulsed with a clinical (PA514) or laboratory (PAO1) strain of P. aeruginosa initiated cross-reacting antibody responses against each other, but not against Escherichia coli and vice versa. These observations may be useful in developing vaccines for infectious diseases, including P. aeruginosa infection.

Infection of endothelium with E1(-)E4(+), but not E1(-)E4(-), adenovirus gene transfer vectors enhances leukocyte adhesion and migration by modulation of ICAM-1, VCAM-1, CD34, and chemokine expression.

Intravascular introduction of replication-deficient adenoviral vectors (Advectors) provides an ideal model of delivery of transgenes for the treatment of various vascular abnormalities. On the basis of the knowledge that Advectors can induce inflammatory responses after intravascular administration, we speculated that cellular activation by Advector infection could directly modulate the endothelial cell (EC) adhesion molecule/chemokine expression repertoire. Infection of human umbilical vein ECs or bone marrow microvascular ECs with an E1(-)E4(+) Advector resulted in the upregulation of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and CD34, but not E-selectin, P-selectin, CD36, CD13, CD44, HLA-DR or PECAM. Upregulation of ICAM-1, VCAM-1, and CD34 was apparent 12 hours after infection and persisted for weeks after infection. Selective induction of adhesion molecules was mediated by the presence of the E4 gene in the Advector, because infection of ECs with an E1(-)E4(-) Advector had no effect on adhesion molecule expression. ECs infected with E1(-)E4(+) Advector, but not those infected with E1(-)E4(-) Advector, supported the adhesion of leukocytes. Monoclonal antibodies to ICAM-1 and VCAM-1 inhibited adhesion of leukocytes to E1(-)E4(+)-infected ECS: Infection of the ECs with E1(-)E4(+) Advector, but not E1(-)E4(-) Advector, resulted in downregulation of expression of chemocytokines, including interleukin-8, MCP-1, RANTES, and GM-CSF. Nonetheless, a large number of leukocytes migrated through ECs infected with E1(-)E4(+), but not those infected with E1(-)E4(l-), in response to exogenous chemokines. These results demonstrate that infection of ECs with E1(-)E4(+) Advectors, but not E1(-)E4(-) Advectors, may directly augment inflammatory responses by upregulating expression of adhesion molecules and enhancing migration through Advector-infected ECs and suggest that E1(-)E4(-) Advectors may be a better choice for gene-transfer strategies directed to the ECS:

Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells.

Tyrosine kinase receptors for angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) are expressed not only by endothelial cells but also by subsets of hematopoietic stem cells (HSCs). To further define their role in the regulation of postnatal hematopoiesis and vasculogenesis, VEGF and Ang-1 plasma levels were elevated by injecting recombinant protein or adenoviral vectors expressing soluble VEGF(165), matrix-bound VEGF(189), or Ang-1 into mice. VEGF(165), but not VEGF(189), induced a rapid mobilization of HSCs and VEGF receptor (VEGFR)2(+) circulating endothelial precursor cells (CEPs). In contrast, Ang-1 induced delayed mobilization of CEPs and HSCs. Combined sustained elevation of Ang-1 and VEGF(165) was associated with an induction of hematopoiesis and increased marrow cellularity followed by proliferation of capillaries and expansion of sinusoidal space. Concomitant to this vascular remodeling, there was a transient depletion of hematopoietic activity in the marrow, which was compensated by an increase in mobilization and recruitment of HSCs and CEPs to the spleen resulting in splenomegaly. Neutralizing monoclonal antibody to VEGFR2 completely inhibited VEGF(165), but not Ang-1-induced mobilization and splenomegaly. These data suggest that temporal and regional activation of VEGF/VEGFR2 and Ang-1/Tie-2 signaling pathways are critical for mobilization and recruitment of HSCs and CEPs and may play a role in the physiology of postnatal angiogenesis and hematopoiesis.

Plasma elevation of stromal cell-derived factor-1 induces mobilization of mature and immature hematopoietic progenitor and stem cells.

The chemokine, stromal cell-derived factor-1 (SDF1), is produced in the bone marrow and has been shown to modulate the homing of stem cells to this site by mediating chemokinesis and chemotaxis. Therefore, it was hypothesized that elevation of SDF1 level in the peripheral circulation would result in mobilization of primitive hematopoietic stem and progenitor cells. SDF1 plasma level was increased by intravenous injection of an adenoviral vector expressing SDF1alpha (AdSDF1) into severe combined immunodeficient mice. This resulted in a 10-fold increase in leukocyte count, a 3-fold increase in platelets, and mobilization of progenitors, including colony-forming units-granulocyte-macrophage to the peripheral circulation. In addition, AdSDF1 induced mobilization of cells with stem cell potential, including colony-forming units in spleen and long-term reconstituting cells. These data demonstrate that overexpression of SDF1 in the peripheral circulation results in the mobilization of hematopoietic cells with repopulating capacity, progenitor cells, and precursor cells. These studies lay the foundation for using SDF1 to induce mobilization of hematopoietic stem and progenitor cells in in vivo studies. (Blood. 2001;97:3354-3360)

Host responses and persistence of vector genome following intrabronchial administration of an E1(-)E3(-) adenovirus gene transfer vector to normal individuals.

Adenovirus (Ad)-mediated gene transfer to the respiratory epithelium of experimental animals and to nasal and airway epithelium of individuals with cystic fibrosis is followed by transient gene expression. Extensive studies in experimental animals are consistent with the concept that local cellular host anti-vector immune responses account for this short-term expression, and systemic and local [lung epithelial lining fluid (ELF)] anti-Ad neutralizing antibodies are generated following Ad vector administration to the respiratory epithelial surface. To determine if this paradigm holds in normal humans, a first-generation Ad vector (Ad(GV)CD.10, an E1(-)E3(-) Ad serotype 5-based vector coding for the Escherichia coli cytosine deaminase gene) was sprayed locally in escalating doses (8 x 10(8)-8 x 10(10) particle units (pu), n = 2/group) into the lung airway epithelium of six normal individuals. Serum, ELF, and endobronchial biopsies were obtained at baseline and at various time points following vector administration. In contrast to the observations in experimental animals in which lung administration of first-generation Ad vectors is followed by strong systemic and local host response, bronchial spray administration of the Ad vector to normal humans showed: (1) minimal inflammation in bronchial biopsies, bronchial brushing, and bronchoalveolar lavage fluid; (2) no blood lymphocyte proliferation in five of six individuals in response to in vitro stimulation with Ad antigens; and (3) no significant increase from baseline in blood or lung ELF anti-Ad neutralizing antibodies. Despite this minimal normal human anti-Ad host response, dose-dependent levels of vector DNA in the airway epithelium were transient. Vector DNA in the targeted airway epithelial cells peaked in a dose-dependent fashion at 0.007 to 1.1 copies/cell at day 7 and declined thereafter, reducing to <10% of peak levels by 2 weeks. These observations demonstrate both the strengths and the limits of using experimental animals to predict human responses to gene transfer vectors. While the transient nature of Ad vector persistence in the airway epithelium is predicted by most experimental animal studies, respiratory epithelial administration of first-generation Ad vectors at doses up to 10(10) pu to airway epithelium of healthy individuals elicits minimal to no detectable systemic and mucosal humoral and cellular immune responses, an observation diametrically opposed to the host responses measured in experimental animals. These findings suggest that, while adaptive anti-Ad immune responses likely play some role in the disappearance of the vector DNA following vector administration to the human lung, other mechanisms may also be involved in the response of humans to Ad gene transfer vectors.

Percutaneous endocardial transfer and expression of genes to the myocardium utilizing fluoroscopic guidance.

Experimental studies indicate that administration of angiogenic proteins or genes by the epicardial or intracoronary route can stimulate development of new collateral vessels and improve myocardial perfusion. An endocardial catheter-based approach to this therapy would obviate the need for surgery, while preserving the effectiveness of direct intramyocardial administration. Fluoroscopic guidance and prototype, preformed, coaxial catheters were used to examine the feasibility of percutaneous catheter-based adenovirus (Ad)-mediated gene transfer and expression in normal swine myocardium. The feasibility of intramyocardial administration (100 microl/injection) of a radiocontrast agent and black tissue dye to all regions of the left ventricle (septum, anterior, lateral, and inferior wall) was confirmed fluoroscopically and on postmortem examination. Injections of replication-deficient adenovirus (10 injections of 10(11) particle units/100 microl each) coding for beta-galactosidase (Adbetagal) or vascular endothelial growth factor (Ad(GV)VEGF121.10) were administered to the left ventricular free wall to examine endocardial based gene transfer and expression. beta-Galactosidase activity was detected by histochemical staining and quantitative assay in targeted regions of the myocardium. Regional VEGF expression was found to be significantly greater in targeted regions (1.3 +/- 0.4 ng/mg protein) as compared with non-targeted regions (0.3 +/- 0.1 ng/mg protein) or regions injected with control (Adbetagal) virus (0.2 +/- 0.03 ng/mg protein, P < 0.001). Catheter-based Ad mediated endocardial gene transfer and expression is feasible using percutaneous, fluoroscopically guided, preformed, coaxial catheters. This approach should be clinically useful to administer angiogenic genes to the ischemic myocardium.

Use of quantitative TaqMan real-time PCR to track the time-dependent distribution of gene transfer vectors in vivo.

To assess the biodistribution and pharmacokinetics of gene transfer vectors, real-time PCR with fluorescent TaqMan chemistry was used to quantify tissue levels of adenovirus gene transfer vectors (Ad) following myocardial administration. After optimizing the detection of the genome of Ad vectors expressing human vascular endothelial growth factor (Ad(GV)VEGF121.10) and Escherichia coli cytosine deaminase (Ad(GV)CD.10), a comparison was made of intramyocardial injection versus intracoronary delivery to the left ventricle of the pig. One hour post-intramyocardial administration, the left ventricular Ad genome level was 6.2 copies per cellular genome, 26-fold higher than the level of 0.24 copies per cellular genome following intracoronary administration. Relative to the vector levels after 1 h, the amount dropped 14- and 5.5-fold by 24 h following intramyocardial and intracoronary administration, respectively. Interestingly, the vector that escaped the left ventricle after intracoronary or intramyocardial administration to pigs was found primarily within the lung, an observation in marked variance to the biodistribution of Ad vector in rodents. In this regard, after intravenous injection to the pig, 90% of the recovered vector was found in the lung, and even after intrahepatic portal vein injection, 55% of the recovered vector was in the lung. These data have important implications regarding the use of experimental animals for safety studies on administration of Ad to humans.

Free cholesterol enhances adenoviral vector gene transfer and expression in CAR-deficient cells.

Efficient adenovirus vector-mediated gene transfer depends on the presence of sufficient amounts of the high-affinity coxsackie-adenovirus (Ad) receptor (CAR) on the surface of the target cell leading to receptor-mediated endocytosis of the vector. The present study evaluates the effect of free cholesterol, a lipid component of endocytic vesicles, on Ad uptake into CAR-deficient cells. Infection in the presence of free cholesterol at its maximum solubility in water led to increased binding, uptake, and expression of Ad in human skin fibroblasts and alveolar macrophages, two primary human cells known to be deficient in CAR. The effect of free cholesterol was maximal at its solubility maximum in aqueous solution. Increase of Ad vector-mediated gene transfer with cholesterol was dependent on the lack of CAR receptor expression on the surface and was diminished by overexpression of CAR in CAR-deficient cells. Cholesterol-mediated increase of Ad-mediated gene expression was dependent on coincubation of both cholesterol and Ad and was not dependent on the cholesterol content of the cell. Increased Ad vector-mediated gene expression in the presence of free cholesterol was also observed in murine skin in vivo. Structural analysis of the Ad-cholesterol mixture showed complexation between Ad particles leading to formation of multivirus aggregates due to hydrophobic interaction. The addition of free cholesterol with Ad vectors may be a simple way to increase Ad-mediated gene transfer to cells that are poor targets due to their lack of a sufficient number of Ad receptors.

Adenoviral vector-mediated gene transfer to primitive human hematopoietic progenitor cells: assessment of transduction and toxicity in long-term culture.

Adenoviral gene transfer to primitive hematopoietic progenitor cells (HPCs) would be useful in gene therapy applications where transient, high-level transgene expression is required. In the present investigations, we have used an adenoviral vector expressing the green fluorescent protein (AdGFP) to quantify transduction of primitive HPCs and assess adenoviral-associated toxicity in long-term culture. Here we show that a cytokine cocktail protects mass populations of CD34(+) cells and primary colony forming unit-cultures (CFU-Cs) from toxicity, enabling transduction of up to 79% of CD34(+) cells. Transduction of CFU-Cs and more primitive HPCs was quantified following fluorescence activated cell sorting for green flourescence protein expression. Our results demonstrate transduction of 45% of primary CFU-Cs, 33% of week-5 cobblestone area forming cells (CAFCs), and 18% of week-5 CFU-Cs. However, AdGFP infection inhibited proliferation of more primitive cells. Although there was no apparent quantitative change in week-5 CAFCs, the clonogenic capacity of week-5 AdGFP-infected cells was reduced by 40% (P <.01) when compared with mock-infected cells. Adenoviral toxicity specifically affected the transduced subset of primitive HPCs. Transduction of primitive cells is therefore probably underestimated by week-5 CFU-Cs and more accurately indicated by week-5 CAFCs. These studies provide the first functional and quantitative evidence of adenoviral transduction of primitive HPCs. However, further investigations will be necessary to overcome adenoviral toxicity toward primitive HPCs before adenoviral vectors can be considered a safe option for gene therapy.

Focal angiogen therapy using intramyocardial delivery of an adenovirus vector coding for vascular endothelial growth factor 121.

BACKGROUND: Adenovirus (Ad) vector-mediated gene therapy strategies have emerged as promising modalities for the "biological revascularization" of tissues. We hypothesized that direct intramyocardial, as opposed to intracoronary, administration of an Ad vector coding for the vascular endothelial growth factor 121 cDNA (Ad(GV)VEGF121.10) would provide highly focal Ad genome levels, and increases in VEGF, ideal for inducing localized therapeutic angiogenesis. METHODS: Persistence and regional distribution of the vector were assessed by TaqMan real-time quantitative polymerase chain reaction technology and enzyme-linked immunosorbent assay, after intramyocardial Ad(GV)VEGF121.10 in the rat, and either intramyocardial or intracoronary (circumflex territory) vector in Yorkshire swine. Based on these results, we assessed the focal nature of the improved cardiac blood flow in a previously reported porcine myocardial ischemia model. RESULTS: Intramyocardial delivery of Ad(GV)VEGF121.10 in the rat resulted in local persistence of the Ad genome that decreased 1,000-fold over 3 weeks, with peak myocardial VEGF expression 24 to 72 h after vector delivery. After intramyocardial Ad(GV)VEGF121.10 in the circumflex distribution of pigs, Ad vector genome and VEGF protein levels were more than 1,000-fold and more than 90-fold higher, respectively, in this distribution than in other myocardial regions. In comparison, intracoronary injection yielded maximum myocardial Ad genome and VEGF levels 33-fold and 9-fold lower, respectively, than that after intramyocardial delivery. Angiograms obtained 28 days after intramyocardial Ad(GV)VEGF121.10 demonstrated rapid circumflex reconstitution via collaterals localized to the region of vector administration. CONCLUSIONS: These studies demonstrate that direct intramyocardial administration of Ad(GV)VEGF121.10 results in focal genome and VEGF levels, including focal angiogenesis, sufficient to normalize blood flow to the ischemic myocardium, findings that are relevant to designing human trials of gene therapy-mediated cardiac angiogenesis.