Human Gene Therapy

Human bocavirus type-1 (HBoV1) has a high tropism for the apical membrane of human airway epithelia. The packaging of a recombinant adeno-associated virus 2 (rAAV2) genome into HBoV1 capsid produces a chimeric vector (rAAV2/HBoV1) that also efficiently transduces human airway epithelia. As such, this vector is attractive for use in gene therapies to treat lung diseases such as cystic fibrosis. However, preclinical development of rAAV2/HBoV1 vectors has been hindered by the fact that humans are the only known host for HBoV1 infection. This study reports that rAAV2/HBoV1 vector is capable of efficiently transducing the lungs of both newborn (3- to 7-day-old) and juvenile (29-day-old) ferrets, predominantly in the distal airways. Analyses of in vivo, ex vivo, and in vitro models of the ferret proximal airway demonstrate that infection of this particular region is less effective than it is in humans. Studies of vector binding and endocytosis in polarized ferret proximal airway epithelial cultures revealed that a lack of effective vector endocytosis is the main cause of inefficient transduction in vitro. While transgene expression declined proportionally with growth of the ferrets following infection at 7 days of age, reinfection of ferrets with rAAV2/HBoV1 at 29 days gave rise to approximately 5-fold higher levels of transduction than observed in naive infected 29-day-old animals. The findings presented here lay the foundation for clinical development of HBoV1 capsid-based vectors for lung gene therapy in cystic fibrosis using ferret models.

Mucopolysaccharidosis type II (MPS II; Hunter syndrome) is a rare X-linked recessive lysosomal disorder caused by defective iduronate-2-sulfatase (IDS), resulting in accumulation of heparan sulfate and dermatan sulfate glycosaminoglycans (GAGs). Enzyme replacement is the only Food and Drug Administration–approved therapy available for MPS II, but it is expensive and does not improve neurologic outcomes in MPS II patients. This study evaluated the effectiveness of adeno-associated virus (AAV) vector encoding human IDS delivered intracerebroventricularly in a murine model of MPS II. Supraphysiological levels of IDS were observed in the circulation (160-fold higher than wild type) for at least 28 weeks post injection and in most tested peripheral organs (up to 270-fold) at 10 months post injection. In contrast, only low levels of IDS were observed (7–40% of wild type) in all areas of the brain. Sustained IDS expression had a profound effect on normalization of GAG in all tested tissues and on prevention of hepatomegaly. Additionally, sustained IDS expression in the central nervous system (CNS) had a prominent effect in preventing neurocognitive deficit in MPS II mice treated at 2 months of age. This study demonstrates that CNS-directed, AAV9 mediated gene transfer is a potentially effective treatment for Hunter syndrome, as well as other monogenic disorders with neurologic involvement.

Previously, the authors developed an adenoviral vector, Ad-PG, where transgene expression is regulated by a p53-responsive promoter. When used to transfer the p53 cDNA, a positive feedback mechanism is established. In the present study, a critical comparison is performed between Ad-PGp53 and AdRGD-PGp53, where the RGD motif was incorporated in the adenoviral fiber protein. AdRGD-PGp53 provided superior transgene expression levels and resulted in the killing of prostate carcinoma cell lines DU145 and PC3. In vitro, this effect was associated with increased production of cytoplasmic and mitochondrial oxidants, DNA damage as revealed by detection of phosphorylated H2AX, as well as cell death consistent with apoptosis. Differential gene expression of key mediators of reactive oxygen species pathways was also observed. Specifically, it was noted that induction of known p53-target genes Sestrin2 and PIG3, as well as a novel target, NOX1, occurred in PC3 cells only when transduced with the improved vector, AdRGD-PGp53. The participation of NOX1 was confirmed upon its inhibition using a specific peptide, resulting in reduced cell death. In situ gene therapy also resulted in significantly improved inhibition of tumor progression consistent with oxidant-induced DNA damage only when treated with the novel AdRGD-PGp53 vector. The study shows that the improved adenovirus overcomes limitations associated with other p53-expressing vectors and induces oxidant-mediating killing, thus supporting its further development for cancer gene therapy.

While therapeutic expression of coagulation factors from adeno-associated virus (AAV) vectors has been successfully achieved in patients with hemophilia, neutralizing antibodies to the vector and inhibitory antibodies to the transgene severely limit efficacy. Indeed, approximately 40% of mice transduced with human factor VIII using the AAV8 serotype developed inhibitory antibodies to factor VIII (FVIII inhibitor), as well as extremely high titers (≥1:500) of neutralizing antibodies to AAV8. To correct hemophilia in these mice, AAV9, a serotype with low in vitro cross-reactivity (≤1:5) to anti-AAV8, was used to deliver mouse-activated factor VII (mFVIIa). It was found that within 6 weeks of systemic administration of 2 × 10¹³ particles/kg of AAV9/mFVIIa, hemophiliac mice with FVIII inhibitors and neutralizing antibodies (NAb) to AAV8 achieved hemostasis comparable to that in wild-type mice, as measured by rotational thromboelastometry. A level of 737 ng/mL mFVIIa was achieved after AAV9/mFVIIa adminstration compared to around 150 ng/mL without vector treatment, and concomitantly prothrombin time was shortened. Tissues collected after intra-articular hemorrhage from FVIII-deficient mice and mice with FVIII inhibitors were scored 4.7 and 5.5, respectively, on a scale of 0–10, indicating significant pathological damage. However, transduction with AAV9/mFVIIa decreased pathology scores to 3.6 and eliminated hemosiderin iron deposition in the synovium in most mice. Collectively, these results suggest that application of alternative serotypes of AAV vector to deliver bypassing reagents has the potential to correct hemophilia and prevent hemoarthrosis, even in the presence of FVIII inhibitor and neutralizing antibodies to AAV.

In advanced and metastatic stages of colorectal cancer (CRC), reduced sensitivity to conventional strategies is still a major obstacle to successful treatments. Decorin is an important regulator in the development and progression of various cancers. To examine if CRC patients have altered decorin levels, expression of decorin and its target genes, Met and vascular endothelial growth factor A (VEGFA), were analyzed in their tumors. Compared to normal tissues, decorin expression was reduced in CRC patients' tumors, while there were increased Met and VEGFA levels. To develop a novel therapy for CRC, rAd.DCN.GM, an oncolytic adenovirus encoding decorin and granulocyte macrophage colony stimulating factor (GM-CSF), has been created. Several therapeutic strategies expressing GM-CSF have been employed in clinical trials for treating metastatic colorectal cancer. In this study, infection of CRC cells with rAd.DCN.GM expressed decorin and GM-CSF, and produced cytotoxicity. In murine CT26 xenografts, rAd.DCN.GM and control adenoviruses were administrated intratumorally on days 7 and 10, and tumor volumes were monitored over time. The study showed that rAd.DCN.GM inhibited the tumor growth and lung metastases significantly. rAd.DCN.GM induced apoptosis, inhibited proliferation, and downregulated angiogenesis and epithelial mesenchymal transition markers in the tumors. On day 12 and day 29, the immune-activation in the peripheral blood, tumors, and spleens were analyzed. rAd.DCN.GM increased CD8⁺ T lymphocytes in the blood, upregulated perforin and granzyme B in the tumors, inhibited transforming growth factor beta expression, and promoted dendritic-cell production in the spleen. In conclusion, rAd.DCN.GM inhibited the tumor growth and metastasis of CT26 tumors, downregulated multiple pro-tumorigenic pathways, and activated antitumor immune responses.

Pulmonary arterial hypertension (PAH) is a progressive disease that culminates in right heart failure and death. Prostacyclin (PGI2) and its derivatives are effective treatments for PAH when administered as continuous parenteral infusions. This treatment paradigm requires medical sophistication, and patients are at risk for complications from an indewelling catheter; drug interruptions may result in rebound pulmonary hypertension and death. We hypothesized that the salivary gland can be repurposed into an endogenous production site for circulating PGI2 through the expression of a fusion protein embodying cyclooxygenase-1 (Cox1) and prostacyclin synthase (PGIS) domains. We utilized ultrasound-assisted gene transfer, a nonviral gene transfer strategy that achieves robust gene transfer to the salivary gland. We initially found that Cox1-PGIS expression in livers of mice using an adenoviral vector dramatically increased circulating PGI2 relative to untreated rats or rats treated with PGIS alone. We then utilized ultrasound-assisted gene transfer to express Cox1-PGIS in the submandibular glands of rats and showed a significant elevation of circulating PGI2 that corresponded to approximately 30% of that seen in humans undergoing intravenous infusion therapy for PAH. These results suggest the feasibility of gene therapy to drive endogenous biosynthesis of PGI2 as a therapeutic strategy for the treatment of PAH.

SNORD44 is a C/D box small nucleolar RNA, and exhibits low expression in breast cancer and head and neck squamous cell carcinoma tissues. Its host gene is growth arrest specific transcript 5 (GAS5), which is a long noncoding RNA. GAS5 is downregulated in colorectal cancer (CRC), and overexpression of GAS5 suppresses cell proliferation. However, the function of SNORD44 in CRC remains largely unknown, and the application of SNORD44 combined with GAS5 in CRC treatment has not been reported. In this study, the expression levels of SNORD44 and GAS5 were measured in CRC tissues by quantitative RT-PCR. The correlation between SNORD44 and GAS5 was evaluated by Pearson correlation analysis. An oncolytic adenovirus expressing SNORD44 and GAS5 (SPDD-UG) was constructed. The biological effects of SPDD-UG were investigated in CRC cell line SW620 and LS174T in vitro and in xenografts. The synergistic effect of rapamycin and SPDD-UG was explored in SW620 and LS174T cells and tumors. We demonstrated that SNORD44 expression level was markedly decreased in CRC tissues and positively correlated with GAS5 expression. SPDD-UG significantly inhibited SW620 and LS174T cell growth and induced cell apoptosis. Intratumoral injection of SPDD-UG significantly suppressed xenografts growth in nude mice. Moreover, the mechanistic target of rapamycin (mTOR) inhibitor, rapamycin, enhanced the antitumor effect through antagonizing the PI3K/Akt pathway activated by SPDD-UG. These results suggest that overexpression of SNORD44 and GAS5 by oncolytic adenovirus provides a promising method for CRC therapy.