Philip W. Zoltick

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.

IL-10 Overexpression Decreases Inflammatory Mediators and Promotes Regenerative Healing in an Adult Model of Scar Formation

Adult wound healing is characterized by an exuberant inflammatory response and scar formation. In contrast, scarless fetal wound healing has diminished inflammation, a lack of fibroplasia, and restoration of normal architecture. We have previously shown that fetal wounds produce less inflammatory cytokines, and the absence of IL-10, an anti-inflammatory cytokine, results in fetal scar formation. We hypothesized that increased IL-10 would decrease inflammation and create an environment conducive for regenerative healing in the adult. To test this hypothesis, a lentiviral vector expressing IL-10 and green fluorescent protein (GFP) (Lenti-IL-10) or GFP alone (Lenti-GFP) was injected at the wound site 48 hours before wounding. We found that both Lenti-IL-10 and Lenti-GFP were expressed in the wounds at 1 and 3 days post wounding. At 3 days, Lenti-IL-10-treated wounds demonstrated decreased inflammation and decreased quantities of all proinflammatory mediators analyzed with statistically different levels of IL-6, monocyte chemoattractant protein-1, and heat-shock protein 47. At 3 weeks, Lenti-GFP wounds demonstrated scar formation. In contrast, wounds injected with Lenti-IL-10 demonstrated decreased inflammation, a lack of abnormal collagen deposition, and restoration of normal dermal architecture. We conclude that lentivirus-mediated overexpression of IL-10 decreases the inflammatory response to injury, creating an environment conducive for regenerative adult wound healing.

Enhanced epithelial gap closure and increased angiogenesis in wounds of diabetic mice treated with adult murine bone marrow stromal progenitor cells.

The direct application of bone marrow (BM) can accelerate the healing of chronic wounds. We hypothesized that this effect is due to the presence of stromal progenitor cells (SPCs) found within whole BM preparations. To test this hypothesis, we isolated adult murine SPCs from whole BM and examined their ability to enhance impaired wound healing compared with ficoll separated BM cells in the diabetic (db/db) mouse model. SPCs significantly enhanced reepithelialization, granulation tissue formation, and neovascularization compared with control wounds treated with BM or PBS alone. Higher frequencies of donor SPC cells compared with donor BM cells were observed in treated wounds at 7 days. Transdifferentiation into GFP‐positive mature endothelial cells was not observed. These observations suggest that SPCs improve wound healing through indirect mechanisms which lead to enhanced vascularization rather than through direct participation and incorporation into tissue. We conclude that topical application of BM‐derived SPCs may represent an effective strategy for the treatment of chronic diabetic wounds.

Molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus, strain A59

Abstract Complementary DNA (cDNA) libraries were constructed representing the genome RNA of the coronavirus mouse hepatitis virus, strain A59 (MHV-A59). From these libraries clones were selected to form a linear map across the entire gene A, the putative viral polymerase gene. This gene is approximately 23 kb in length, considerably larger than earlier estimates. Sequence analysis of the U terminal region of the genome indicates the presence of the 66-nucleotide leader that is found on all mRNAs. Secondary structure analysis of the 5′terminal region suggests that transcription of leader terminates in the region of nucleotide 66. The sequence of the first 2000 nucleotides is very similar to that reported for the closely related JHM strain of MHV and potentially encodes p28, a basic protein thought to be a component of the viral polymerase (L. Soe, C. K. Shieh, S. Baker, M. F. Chang, and M. M. C. Lai, 1987, J. Virol., 61, 3968–3976). Gene A contains two of the consensus sequences found in intergenic regions. One is adjacent to the 5′ leader sequence and the other is upstream from the initiation codon for translation of gene B.

Permissive environment in postnatal wounds induced by adenoviral-mediated overexpression of the anti-inflammatory cytokine interleukin-10 prevents scar formation

Wound healing in the mid‐gestation fetus is scarless with minimal inflammation and a unique extracellular matrix. We have previously documented the relative lack of inflammatory cytokines in this environment. We demonstrate that interleukin (IL)‐10 is highly expressed in mid‐gestation human fetal skin but is absent in postnatal human skin. We hypothesize that overexpression of IL‐10 in postnatal skin may replicate a permissive environment for scarless healing. To study the mechanism underlying this process we performed immunohistochemistry for IL‐10 in human mid‐gestation fetal and postnatal skin. We also determined if adenoviral‐mediated overexpression of IL‐10 could allow for scarless wound healing in a murine incisional wound model. Wounds were analyzed at 1–90 days postwounding for effects on scar formation, inflammatory response, and biomechanical properties. Ad‐IL‐10 reconstitutes a permissive environment for scarless healing as shown by reconstitution of a normal dermal reticular collagen pattern and distribution of dermal elements. Compared with controls, Ad‐IL‐10 treated wounds showed reduced inflammatory response and no difference in biomechanical parameters. Therefore, overexpression of IL‐10 in postnatal wounds results in a permissive environment for scarless wound repair, possibly by replicating a fetal wound environment.

Intracellular processing of the N-terminal ORF 1a proteins of the coronavirus MHV-A59 requires multiple proteolytic events

Abstract Several polypeptide products of MHV-A59 ORF 1a were characterized in MHV-A59 infected DBT cells, using antisera directed against fusionn roteins encoded in the first 6.5 kb of ORF1a. These included the previously identified N-terminal ORF 1a product, p28, as well as 290-, 240-, and 50-kDa polypeptides. P28 was always detected as a discrete band without larger precursors, suggesting rapid cleavage of p28 immediately after its synthesis. Once p28 was cleaved there was little degradation of the protein over a 2-hr period. The intracellular cleavage of p28 was not inhibited by the protease inhibitor leupeptin, in contrast to results obtained during in vitro translation of genome RNA (Denison and Perlman, 1986). These data suggest that different protease activities may be responsible for the cleavage of p28 in vitro and in vivo. The 290-kDa protein was an intermediate cleavage product derived from a precursor of greater than 400 kDa. The 290-kDa product was subsequently cleaved into secondary products of 50 and 240 kDa. The intracellular cleavage of the 290-kDa polypeptide was inhibited by leupeptin at concentrations which did not inhibit the early cleavage of p28 or the cleavage of the 290-kDa product from its larger polyprotein precursor. In the presence of zinc chloride, a product of >320 kDa was detected, which appears to incorporate p28 at its amino terminus. This suggests that at least two protease activities may be necessary for processing of ORF1a proteins, one of which cleaves p28 and is sensitive to zinc chloride but resistant to leupeptin, and the other which cleaves the 290-kDa precursor and is sensitive to both inhibitors. Both the 290- and 240-kDa proteins should contain sequences predicted to encode two papain-like protease activities.

Cystic Adenomatoid Malformations Are Induced by Localized FGF10 Overexpression in Fetal Rat Lung

Fibroblast growth factor-10 (FGF10) is a mesenchymal growth factor, involved in epithelial and mesenchymal interactions during lung branching morphogenesis. In the present work, FGF10 overexpression was transiently induced in a temporally and spatially restricted manner, during the pseudoglandular or canalicular stages of rat lung development, by trans-uterine ultrasound-guided intraparenchymal microinjections of adenoviral vector encoding the rfgf10 transgene. The morphologic and histologic classification of the resulting malformations were dependent upon developmental stage and location. Overexpression of FGF10 restricted to the proximal tracheobronchial tree during the pseudoglandular phase resulted in large cysts lined by tall columnar epithelium composed primarily of Clara cells with a paucity of Type II pneumocytes, resembling bronchiolar type epithelium. In contrast, FGF10 overexpression in the distal lung parenchyma during the canalicular phase resulted in small cysts lined by cuboidal epithelial cells composed of primarily Type II pneumocytes resembling acinar epithelial differentiation. The cystic malformations induced by FGF10 overexpression appear to closely recapitulate the morphology and histology of the spectrum of human congenital cystic adenomatoid malformation (CCAM). These findings support a role for FGF10 in the induction of human CCAM and provide further mechanistic insight into the role of FGF10 in normal and abnormal lung development.

Maternal alloantibodies induce a postnatal immune response that limits engraftment following in utero hematopoietic cell transplantation in mice

The lack of fetal immune responses to foreign antigens, i.e., fetal immunologic tolerance, is the most compelling rationale for prenatal stem cell and gene therapy. However, the frequency of engraftment following in utero hematopoietic cell transplantation (IUHCT) in the murine model is reduced in allogeneic, compared with congenic, recipients. This observation supports the existence of an immune barrier to fetal transplantation and challenges the classic assumptions of fetal tolerance. Here, we present evidence that supports the presence of an adaptive immune response in murine recipients of IUHCT that failed to maintain engraftment. However, when IUHCT recipients were fostered by surrogate mothers, they all maintained long-term chimerism. Furthermore, we have demonstrated that the cells responsible for rejection of the graft were recipient in origin. Our observations suggest a mechanism by which IUHCT-dependent sensitization of the maternal immune system and the subsequent transmission of maternal alloantibodies to pups through breast milk induces a postnatal adaptive immune response in the recipient, which, in turn, results in the ablation of engraftment after IUHCT. Finally, we showed that non-fostered pups that maintained their chimerism had higher levels of Tregs as well as a more suppressive Treg phenotype than their non-chimeric, non-fostered siblings. This study resolves the apparent contradiction of induction of an adaptive immune response in the pre-immune fetus and confirms the potential of actively acquired tolerance to facilitate prenatal therapeutic applications.

Correction of ADAMTS13 Deficiency by In Utero Gene Transfer of Lentiviral Vector encoding ADAMTS13 Genes

Deficiency of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS13) results in thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective treatment to date. We show in this study that an administration of a self-inactivating lentiviral vector encoding human full-length ADAMTS13 and a variant truncated after the spacer domain (MDTCS) in mice by in utero injection at embryonic days 8 and 14 resulted in detectable plasma proteolytic activity (approximately 5-70%), which persisted for the length of the study (up to 24 weeks). Intravascular injection via a vitelline vein at E14 was associated with significantly lower rate of fetal loss than intra-amniotic injection, suggesting that the administration of vector at E14 may be a preferred gestational age for vector delivery. The mice expressing ADAMTS13 and MDTCS exhibited reduced sizes of von Willebrand factor (vWF) compared to the Adamts13(-/-) mice expressing enhanced green fluorescent protein (eGFP). Moreover, the mice expressing both ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time as compared to the Adamts13(-/-) expressing eGFP. The data demonstrate the successful correction of the prothrombotic phenotypes in Adamts13(-/-) mice by a single in utero injection of lentiviral vectors encoding human ADAMTS13 genes, providing the basis for developing a gene therapy for hereditary TTP in humans.

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.