Joseph V. Hughes

A Pilot Study of In Vivo Liver-Directed Gene Transfer with an Adenoviral Vector in Partial Ornithine Transcarbamylase Deficiency

Ornithine transcarbamylase deficiency (OTCD) is an inborn error of urea synthesis that has been considered as a model for liver-directed gene therapy. Current treatment has failed to avert a high mortality or morbidity from hyperammonemic coma. Restoration of enzyme activity in the liver should suffice to normalize metabolism. An E1- and E4-deleted vector based on adenovirus type 5 and containing human OTC cDNA was infused into the right hepatic artery in adults with partial OTCD. Six cohorts of three or four subjects received 1/2 log-increasing doses of vector from 2 x 10(9) to 6 x 10(11) particles/kg. This paper describes the experience in all but the last subject, who experienced lethal complications. Adverse effects included a flu-like episode and a transient rise in temperature, hepatic transaminases, thrombocytopenia, and hypophosphatemia. Humoral responses to the vector were seen in all research subjects and a proliferative cellular response to the vector developed in apparently naive subjects. In situ hybridization studies showed transgene expression in hepatocytes of 7 of 17 subjects. Three of 11 subjects with symptoms related to OTCD showed modest increases in urea cycle metabolic activity that were not statistically significant. The low levels of gene transfer detected in this trial suggest that at the doses tested, significant metabolic correction did not occur.

Humoral Immunity to Adeno-Associated Virus Type 2 Vectors following Administration to Murine and Nonhuman Primate Muscle

ABSTRACT Adeno-associated virus (AAV) is being developed as a vector capable of conferring long-term gene expression, which is useful in the treatment of chronic diseases. In most therapeutic applications, it is necessary to readminister the vector. This study characterizes the humoral immune response to AAV capsid proteins following intramuscular injection and its impact on vector readministration. Studies of mice and rhesus monkeys demonstrated the formation of neutralizing antibodies to AAV capsid proteins that persisted for over 1 year and then diminished, but this did not prevent the efficacy of vector readministration. More-detailed studies strongly suggested that the B-cell response was T cell dependent. This was further evaluated with a blocking antibody to human CD4, primatized for clinical trials, in a biologically compatible mouse in which the endogenous murine CD4 gene was functionally replaced with the human counterpart. Transient pharmacologic inhibition of CD4 T cells with CD4 antibody prevented an antivector response long after the effects of the CD4 antibody diminished; readministration of vector without diminution of gene expression was possible. Our studies suggest that truly durable transgene expression (i.e., prolonged genetic engraftment together with vector readministration) is possible with AAV in skeletal muscle, although it will be necessary to transiently inhibit CD4 T-cell function to avoid the activation of memory B cells.

A phase I study of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator gene to a lung segment of individuals with cystic fibrosis

A third-generation adenoviral vector containing recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) gene was delivered by bronchoscope in escalating doses to the conducting airway of 11 volunteers with cystic fibrosis. Assessments of dose-limiting toxicity (DLT), efficiency of gene transfer, and cell-mediated and humoral immune responses to vector administration were performed. DLT, manifest by flulike symptoms and transient radiographic infiltrates, was seen at 2.1 x 10(11) total viral particles. A highly specific assay for gene transfer was developed using in situ hybridization with an oligoprobe against unique vector sequence. Detectable gene transfer was observed in harvested bronchial epithelial cells (<1%) 4 days after vector instillation, which diminished to undetectable levels by day 43. Adenovirus-specific cell-mediated T cells were induced in most subjects, although only mild increases in systemic humoral immune response were observed. These results demonstrate that gene transfer to epithelium of the lower respiratory tract can be achieved in humans with adenoviral vectors but that efficiency is low and of short duration in the native CF airway.

Readministration of Adenovirus Vector in Nonhuman Primate Lungs by Blockade of CD40-CD40 Ligand Interactions

ABSTRACT The interaction between CD40 on B cells and CD40 ligand (CD40L) on activated T cells is important for B-cell differentiation in T-cell-dependent humoral responses. We have extended our previous murine studies of CD40-CD40L in adenoviral vector-mediated immune responses to rhesus monkeys. Primary immune responses to adenoviral vectors and the ability to readminister vector were studied in rhesus monkeys in the presence or absence of a transient treatment with a humanized anti-CD40 ligand antibody (hu5C8). Adult animals were treated with hu5C8 at the time vector was instilled into the lung. Immunological analyses demonstrated suppression of adenovirus-induced lymphoproliferation and cytokine responses (interleukin-2 [IL-2], gamma interferon, IL-4, and IL-10) in hu5C8-treated animals. Animals treated with hu5C8 secreted adenovirus-specific immunoglobulin M (IgM) levels comparable to control animals, but did not secrete IgA or develop neutralizing antibodies; consequently, the animals could be readministered with adenovirus vector expressing alkaline phosphatase. A second study was designed to examine the long-term effects on immune functions of a short course of hu5C8. Acute hu5C8 treatment resulted in significant and prolonged inhibition of the adenovirus-specific humoral response well beyond the time hu5C8 effects were no longer significant. These studies demonstrate the potential of hu5C8 as an immunomodulatory regimen to enable administration of adenoviral vectors, and they advocate testing this model in humans.

Evaluation of an E1E4-Deleted Adenovirus Expressing the Herpes Simplex Thymidine Kinase Suicide Gene in Cancer Gene Therapy

Studies with first-generation adenoviral vectors have uncovered limitations that include finite transgene persistence, potential hepatotoxicity, and contamination with replication-competent adenovirus (RCA). To address these limitations within the context of cancer suicide gene therapy, a new adenoviral vector was developed containing the herpes simplex virus type 1 thymidine kinase (HSV tk) gene inserted in the E1 region of a recombinant vector containing deletions in the E1 and E4 regions of the Ad5 genome. The HSV tk minigene was placed under transcriptional control of a Rous sarcoma virus (RSV) promoter. This new E1E4-deleted vector was compared with the first-generation E1E3-deleted Ad.RSVtk vector. Generation of replication-competent adenovirus during production was eliminated. Using semiquantitative immunoblotting, the two vectors produced equivalent amounts of the expected 44-kDa tk-encoded protein in three different cell lines tested. The ability of the E1E4-deleted vector to sensitize tumor cells to ganciclovir (GCV) using in vitro assays and mixing studies was comparable to that of the E1E3-deleted vector. In vivo bystander effects were investigated using mixing studies in a syngeneic flank tumor model and demonstrated no difference between vectors in either immunocompetent or immunodeficient mice. To test the efficiency of these vectors in treating tumors in clinically relevant models, virus was injected intraperitoneally into tumor-bearing SCID mice and intrapleurally in a syngeneic rat mesothelioma model. After treatment of animals with ganciclovir, both vectors were roughly equivalent in their ability to increase mean survival (from approximately 40 to approximately 70 days) and markedly reduce tumor burden. Finally, formal toxicology studies were performed and showed similar amounts of local inflammation without systemic toxicity. In summary, this series of in vitro and in vivo experiments indicates that the performance of the recombinant E1E4-deleted adenoviral vector was virtually identical to that of the E1E3-deleted vector. Since the E1E4 vector has a much lower rate of recombination during production and has been shown to be less hepatotoxic in animal models, this new vector should prove superior to the first-generation Ad.HSVtk vectors in clinical cancer gene therapy trials.

Biology of E1-Deleted Adenovirus Vectors in Nonhuman Primate Muscle

ABSTRACT Adenovirus vectors have been studied as vehicles for gene transfer to skeletal muscle, an attractive target for gene therapies for inherited and acquired diseases. In this setting, immune responses to viral proteins and/or transgene products cause inflammation and lead to loss of transgene expression. A few studies in murine models have suggested that the destructive cell-mediated immune response to virally encoded proteins of E1-deleted adenovirus may not contribute to the elimination of transgene-expressing cells. However, the impact of immune responses following intramuscular administration of adenovirus vectors on transgene stability has not been elucidated in larger animal models such as nonhuman primates. Here we demonstrate that intramuscular administration of E1-deleted adenovirus vector expressing rhesus monkey erythropoietin or growth hormone to rhesus monkeys results in generation of a Th1-dependent cytotoxic T-cell response to adenovirus proteins. Transgene expression dropped significantly over time but was still detectable in some animals after 6 months. Systemic levels of adenovirus-specific neutralizing antibodies were generated, which blocked vector readministration. These studies indicate that the cellular and humoral immune response generated to adenovirus proteins, in the context of transgenes encoding self-proteins, hinders long-term transgene expression and readministration with first-generation vectors.

Alterations in Peptide Structure of Vesicular Stomatitis Virus Mutant and its Central Nervous System Isolate

Gradient SDS-polyacrylamide gel electrophoresis (PAGE) and proteolytic digestions were utilized to examine the virion proteins of two isolates of wild-type vesicular stomatitis virus (WT-VSV), WTATCC from the American Type Culture Collection and WTGL and Glasgow, as well as temperature-sensitive (ts) mutant ts G31 and a central nervous system (CNS) isolate of ts G31 designated ts G31BP. The WTATCC M protein differed in electrophoretic mobility and in its tryptic or chymotryptic peptide maps from the 125I-labelled M proteins in WTGL, ts G31 or ts G31BP. The M protein in the latter three viruses appeared identical using either tryptic or chymotryptic digestion procedures; however, limited digestion with V8 protease revealed a difference between the M protein of ts G31 and both WTGL and ts G31BP M proteins. The L, NS and G proteins all had identical tryptic and chymotryptic peptide maps in WTGL, ts G31 and ts G31BP virions. The N protein, however, was demonstrated to be distinctly different in the WTGL virion when compared with the ts G31 (or ts G31BP) virion by its tryptic peptide map. In addition, limited proteolytic digestion of the 125I-labelled N proteins revealed a different peptide structure in ts G31BP compared to N proteins of ts G31 or WTGL. The altered N protein in the CNS isolate, ts G31BP, is discussed in terms of its altered in vivo phenotype of labile viral RNA, and its potential role in the unique CNS disease associated with this virus.

RNA degradation defect in central nervous system isolates of vesicular stomatitis virus.

Six temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV) isolated from the central nervous system (CNS) following injection with ts G31 (III) all possessed a post-transcriptional defect, not found in the initial virus, that affects the stability of viral RNA transcripts. Examination of viral RNA metabolism in mouse neuroblastoma (N-18) cells revealed that RNA synthesis of the CNS isolates was decreased considerably at elevated temperatures (up to 80 or 90% at 39 degrees C). In addition, analysis of the RNA transcripts suggested that little if any normal-sized transcripts were made in cells infected with these CNS isolates at either 37 degrees C or 39 degrees C. The RNA deficiencies did not appear to be the result of a temperature-sensitive lability of virion transcriptase as examined by in vitro transcriptase assays. However, when N-18 cells infected with one of the CNS isolates, ts G31 BP, were first preincubated at the permissive temperature of 31 degrees C for 3 h and then shifted to 39 degrees C, RNA synthesis proceeded at a rate comparable to that of 31 degrees C. The viral mRNA species synthesized following the temperature shift also contained normal sized tracts of poly(A) RNA, suggesting that neither the viral transcriptase nor its polyadenylate synthetase was thermally labile. However, for any of the six CNS isolates, all species of viral RNA synthesized in cells that were first preincubated at 31 degrees C degraded rapidly when the cells were shifted to 39 degrees C. In contrast little or no RNA degradation of either 42S progeny RNA or mRNA species was detected in the wild-type VSV, ts G31 or three other VSV mutants that are defective in some aspect of viral RNA metabolism: [ts G11 (I), ts G22 (II), ts G41 (IV)]. The apparent phenotype alteration in the stability of viral RNA in all of these CNS isolates is discussed in terms of the possible genotypic changes that may have occurred as well a the unique CNS disease that accompanies infection by these viruses.