Long-term correction of hemophilia A mice following lentiviral mediated delivery of an optimized canine factor VIII gene.

Current therapies for hemophilia A include frequent prophylactic or on-demand intravenous factor treatments which are costly, inconvenient and may lead to inhibitor formation. Viral vector delivery of factor VIII (FVIII) cDNA has the potential to alleviate the debilitating clotting defects. Lentiviral-based vectors delivered to murine models of hemophilia A mediate phenotypic correction. However, a limitation of lentiviral-mediated FVIII delivery is inefficient transduction of target cells. Here, we engineer a feline immunodeficiency virus (FIV) -based lentiviral vector pseudotyped with the baculovirus GP64 envelope glycoprotein to mediate efficient gene transfer to mouse hepatocytes. In anticipation of future studies in FVIII-deficient dogs, we investigated the efficacy of FIV-delivered canine FVIII (cFVIII). Codon-optimization of the cFVIII sequence increased activity and decreased blood loss as compared to the native sequence. Further, we compared a standard B-domain deleted FVIII cDNA to a cDNA including 256 amino acids of the B-domain with 11 potential asparagine-linked oligosaccharide linkages. Restoring a partial B-domain resulted in modest reduction of endoplasmic reticulum (ER) stress markers. Importantly, our optimized vectors achieved wild-type levels of phenotypic correction with minimal inhibitor formation. These studies provide insights into optimal design of a therapeutically relevant gene therapy vector for a devastating bleeding disorder.

Novel GP64 envelope variants for improved delivery to human airway epithelial cells.

Lentiviral vectors pseudotyped with the baculovirus envelope protein GP64 transduce primary cultures of human airway epithelia (HAE) at their apical surface. Our goal in this study was to harness a directed evolution approach to develop a novel envelope glycoprotein with increased transduction properties for HAE. Using error-prone PCR, a library of GP64 mutants was generated and used to prepare a diverse pool of lentiviral virions pseudotyped with GP64 variants. The library was serially passaged on HAE and three GP64 mutations were recovered. Single-, double- and the triple-combination mutant envelope glycoproteins were compared with wild-type GP64 for their ability to transduce HAE. Our results suggest that lentiviral vectors pseudotyped with evolved GP64 transduced HAE with greater efficiency than wild-type GP64. This effect was not observed in primary cultures of porcine airway epithelial cells, suggesting that the directed evolution protocol was species specific. In summary, our studies indicate that serial passage of a GP64 mutant library yielded specific variants with improved HAE cell tropism, yielding tools with the potential to improve the success of gene therapy for airway diseases.

Progress and prospects: prospects of repeated pulmonary administration of viral vectors.

Pulmonary gene therapy may ultimately cure diseases such as cystic fibrosis, alpha1-antitrypsin deficiency, lung cancer and pulmonary hypertension. Efficient expression of delivered genes in target cell types is essential for the achievement of this goal. To this end, re-administration of viral vectors may be required (1) to increase the percentage of transduced airway epithelial cells, (2) to direct gene transfer to individual lobes during successive delivery sessions or (3) to boost attenuated expression over time. Immune responses to viral proteins or viral-encoded proteins are the greatest barrier to repeated vector administration.

Gene therapy progress and prospects: development of improved lentiviral and retroviral vectors--design, biosafety, and production.

Replication defective vectors derived from simple retroviruses or the more complex genomes of lentiviruses continue to offer the advantages of long-term expression, cell and tissue specific tropism, and large packaging capacity for the delivery of therapeutic genes. The occurrence of adverse events caused by insertional mutagenesis in three patients in a gene therapy trial for X-linked SCID emphasizes the potential for problems in translating this approach to the clinic. Several genome-wide studies of retroviral integration are now providing novel insights into the integration site preferences of different vector classes. We review recent developments in vector design, integration, biosafety, and production.

Identification of three human renin mRNA isoforms from alternative tissue-specific transcriptional initiation.

We have reported that mice transgenic for 140- and 160-kb P1 phage artificial chromosomes (PACs) containing the human renin gene express the gene in a highly tissue-restricted and regulated manner. Herein, we demonstrate that the transgene is also expressed appropriately throughout development. In the course of this investigation, we identified the existence of three transcriptional isoforms of human renin mRNA derived from the utilization of alternative transcription start sites. The first isoform is the kidney-specific isoform, which utilizes the classic renin promoter. The second is a brain-specific isoform, which when previously identified in rats and mice was due to a transcription initiation site within intron A. However, the start site in the human gene resides approximately 1,325 bp upstream of the classic promoter and encodes a new exon 1 (termed exon 1b) that splices directly to exon 2. The third isoform is lung specific and is due to transcriptional initiation 79 bp directly upstream of exon 2, fusing additional DNA within intron A (termed exon 1c) directly to exon 2 without splicing. Importantly, the alternative first exons observed in the PAC transgenic mice were identical to those used to transcribe renin in human fetal kidney, brain, and lung, suggesting these sites are bona fide isoforms of human renin mRNA and not artifacts of transgenesis. Moreover, the subtle differences in tissue-specific transcriptional initiation observed in the renin gene of rats and humans can be faithfully and accurately emulated in a transgenic model.

Transgenic models as tools for studying the regulation of human renin expression.

Transgenic mice and rats have become popular tools to study the regulation of gene expression and the consequences of protein over-production. Over the past decade, numerous transgenic models have been developed to study the mechanisms of human renin gene expression and the participation of the renin-angiotensin system in the development of hypertension. Herein we will provide an overview of what has been learned from the use of transgenic models for studying the human renin gene.

Development of retroviral vectors for gene transfer to airway epithelia.

Retroviral vectors offer several potential advantages for attaining persistent expression of a therapeutic gene in airway epithelia for diseases such as cystic fibrosis. However, several problems have limited their application. Developments in vector production and the advent of lentiviral vectors have increased the investigation of recombinant retrovirus for gene transfer to airway epithelia. In addition, an improved understanding of some of the barriers limiting gene transfer has led to increased transduction efficiencies. The development of novel vector formulations and the use of new envelope pseudotypes are examples of recent findings that are leading to advances in this field.

Highly regulated cell type-restricted expression of human renin in mice containing 140- or 160-kilobase pair P1 phage artificial chromosome transgenes.

We generated transgenic mice with two P1 artificial chromosomes, each containing the human renin (HREN) gene and extending to -35 and -75 kilobase pairs, respectively. HREN protein production was restricted to juxtaglomerular cells of the kidney, and its expression was tightly regulated by angiotensin II and sodium. The magnitude of the up- and down-regulation in HREN mRNA caused by the stimuli tested was identical to the endogenous renin gene, suggesting tight physiological regulation. P1 artificial chromosome mice were mated with transgenic mice overexpressing human angiotensinogen to determine if there was a chronic compensatory down-regulation of the transgene. Despite a 3-fold down-regulation of HREN mRNA, plasma angiotensin II and blood pressure was modestly elevated in the double transgenic mice. Nevertheless, this elevation was significantly less than a different double transgenic model containing a poorly regulated HREN transgene. The increase in blood pressure, despite the decrease in HREN mRNA, suggests that the HREN gene can partially, but not completely, compensate for excess circulating angiotensinogen. These data suggest the possibility that increases in circulating or tissue angiotensinogen may cause an increase in blood pressure in humans, even in the presence of a functionally active servo-mechanism to down-regulate HREN expression.

JG cell expression and partial regulation of a human renin genomic transgene driven by a minimal renin promoter.

In the kidney, renin gene expression is exquisitely localized to the juxtaglomerular (JG) cells lining the afferent arteriole, having the capacity to regulate renin synthesis in response to a variety of physiological cues. We investigated human renin gene expression in transgenic mice containing a genomic construct driven by 149 bp of its proximal promoter to elucidate whether this was sufficient to confer JG-specific expression. Whereas human renin mRNA was permissively expressed in most tissues, the transgene was expressed mainly in JG cells in the kidney. Active human renin and human prorenin were found in the systemic circulation at levels consistent with previous transgenic models. Remarkably, two lines displayed an appropriate upregulation of transgene mRNA in response to angiotensin-converting enzyme inhibition, and two lines exhibited a downregulation of transgene mRNA in response to subpressor and pressor doses of ANG II. Our results suggest that 149 bp of the human renin proximal promoter, in a context of a genomic construct, are sufficient to confer human renin expression in renal JG cells and at least some aspects of appropriate regulation.

Human renin mRNA stability is increased in response to cAMP in Calu-6 cells.

The human carcinoma-derived cell line Calu-6 has previously been demonstrated to endogenously express human renin (hREN) mRNA and to markedly increase steady-state hREN mRNA levels (100-fold after 24 hours) in response to analogues of cAMP and postreceptor activators of adenylyl cyclase such as forskolin. However, both transfection analysis using hREN promoter-reporter constructs and nuclear run-on experiments suggest that transcriptional activity alone cannot account for this level of induction. We performed primer extension, reverse transcription-polymerase chain reaction, and 3' rapid amplification of cDNA ends to compare hREN mRNA between unstimulated and forskolin-stimulated cells. We demonstrate that hREN mRNA is identical under both conditions with respect to (1) utilization of the appropriate transcription start site, (2) processing of renin mRNA, and (3) utilization of the proper polyadenylation site and length of the poly-A tail. To address the mechanism of induction caused by cAMP, we used transcriptional inhibition and measured decay of hREN mRNA before and after forskolin or phorbol ester treatment. Experiments with both actinomycin D and 5, 6-dichlororibofuranosylbenzimidazole (DRB) showed that forskolin treatment markedly stabilized hREN mRNA in Calu-6 cells. A 2.3-fold increase in hREN mRNA half-life was also observed after treatment of Calu-6 cells with phorbol ester. Experiments with DRB demonstrated a similar robust stabilization of hREN mRNA after forskolin and phorbol ester treatment. These data demonstrate that the induction in hREN mRNA in response to both cAMP and phorbol ester occurs by a mechanism involving a posttranscriptional component.