Transduction, tropism, and biodistribution of AAV vectors in the lacrimal gland.

PURPOSE: The lacrimal gland (LG) delivers defensive and metabolic factors to the ocular surface. These functions may be disrupted in several diseases, and for most of them there is no cure. The aim of this study is to investigate conditions and limitations for using adeno-associated virus (AAV) vectors as gene transfer agents to LG. METHODS: Eight-week-old Balb/c mice were used to investigate route, gene expression, and time course of AAV gene vector transfer to LG. AAV vectors encoding firefly luciferase were administered to the LG and luciferase expression was evaluated in vivo by immunohistochemistry. Ocular surface and neutralizing antibodies were also evaluated. RESULTS: The present work revealed that AAV vectors are able to delivery DNA to the LGs of mice. Direct injection had the highest level of transduction, and topical ocular drops the lowest. Overall, the AAV strain with highest transduction activity as measured by both luminescence and immunohistochemistry was AAV9, followed by AAV 5w8 and AAV5. Transduction was not different between sexes, could be detected as soon as 24 hours after injection, and lasted for at least 30 days (study termination). No tissue damage was observed when compared with controls. All vectors with detectable LG transduction induced neutralizing antibodies. CONCLUSIONS: LG gene delivery by AAV vectors appears to be both safe and well tolerated. The choice of vector influences both the overall transduction activity, as well as the spread of vector to other organs. This work supports the use of AAV-mediated gene therapy for dry eye.

Intranasal administration of adeno-associated virus type 12 (AAV12) leads to transduction of the nasal epithelia and can initiate transgene-specific immune response.

A critical aspect in defining the utility of a vector for gene therapy applications is the cell tropism and biodistribution of the vector. Adeno-associated virus type 12 (AAV12) has several unique biological and immunological properties that could be exploited for gene therapy purposes, including a unique cell surface receptor, transduction of epithelial cells, and limited neutralization by pooled human antibodies. However, little is known about its cell tropism and biodistribution in vivo. In vivo biodistribution studies with AAV12 vectors encoding a cytomegalovirus promoted luciferase transgene indicated preferential transduction of the nasal epithelia which was not observed with AAV2-based vectors. Expression peaked 2 weeks postadministration, before decreasing to a persistent level. The level of neutralizing antibodies (Nab) induced was sevenfold lower for AAV12 than for AAV2, an advantage for use in repeat administration. Furthermore, vectors encoding influenza A nucleoprotein (NP), an antigen which has previously been shown to induce immune protection against challenge, resulted in generation of both anti-A/NP antibodies and lung anti-A/NP T cells. Our findings suggest further evaluation of AAV12 as a vector for gene therapy and as a potential nasal vaccine.

T-cell immunoglobulin and mucin domain 1 (TIM-1) is a receptor for Zaire Ebolavirus and Lake Victoria Marburgvirus.

The glycoproteins (GP) of enveloped viruses facilitate entry into the host cell by interacting with specific cellular receptors. Despite extensive study, a cellular receptor for the deadly filoviruses Ebolavirus and Marburgvirus has yet to be identified and characterized. Here, we show that T-cell Ig and mucin domain 1 (TIM-1) binds to the receptor binding domain of the Zaire Ebola virus (EBOV) glycoprotein, and ectopic TIM-1 expression in poorly permissive cells enhances EBOV infection by 10- to 30-fold. Conversely, reduction of cell-surface expression of TIM-1 by RNAi decreased infection of highly permissive Vero cells. TIM-1 expression within the human body is broader than previously appreciated, with expression on mucosal epithelia from the trachea, cornea, and conjunctiva--tissues believed to be important during in vivo transmission of filoviruses. Recognition that TIM-1 serves as a receptor for filoviruses on these mucosal epithelial surfaces provides a mechanistic understanding of routes of entry into the human body via inhalation of aerosol particles or hand-to-eye contact. ARD5, a monoclonal antibody against the IgV domain of TIM-1, blocked EBOV binding and infection, suggesting that antibodies or small molecules directed against this cellular receptor may provide effective filovirus antivirals.

Tyrosine kinase receptor Axl enhances entry of Zaire ebolavirus without direct interactions with the viral glycoprotein.

In a bioinformatics-based screen for cellular genes that enhance Zaire ebolavirus (ZEBOV) transduction, AXL mRNA expression strongly correlated with ZEBOV infection. A series of cell lines and primary cells were identified that require Axl for optimal ZEBOV entry. Using one of these cell lines, we identified ZEBOV entry events that are Axl-dependent. Interactions between ZEBOV-GP and the Axl ectodomain were not detected in immunoprecipitations and reduction of surface-expressed Axl by RNAi did not alter ZEBOV-GP binding, providing evidence that Axl does not serve as a receptor for the virus. However, RNAi knock down of Axl reduced ZEBOV pseudovirion internalization and α-Axl antisera inhibited pseudovirion fusion with cellular membranes. Consistent with the importance of Axl for ZEBOV transduction, Axl transiently co-localized on the surface of cells with ZEBOV virus particles and was internalized during virion transduction. In total, these findings indicate that endosomal uptake of filoviruses is facilitated by Axl.

Rho GTPases modulate entry of Ebola virus and vesicular stomatitis virus pseudotyped vectors.

To explore mechanisms of entry for Ebola virus (EBOV) glycoprotein (GP) pseudotyped virions, we used comparative gene analysis to identify genes whose expression correlated with viral transduction. Candidate genes were identified by using EBOV GP pseudotyped virions to transduce human tumor cell lines that had previously been characterized by cDNA microarray. Transduction profiles for each of these cell lines were generated, and a significant positive correlation was observed between RhoC expression and permissivity for EBOV vector transduction. This correlation was not specific for EBOV vector alone as RhoC also correlated highly with transduction of vesicular stomatitis virus GP (VSVG) pseudotyped vector. Levels of RhoC protein in EBOV and VSV permissive and nonpermissive cells were consistent with the cDNA gene array findings. Additionally, vector transduction was elevated in cells that expressed high levels of endogenous RhoC but not RhoA. RhoB and RhoC overexpression significantly increased EBOV GP and VSVG pseudotyped vector transduction but had minimal effect on human immunodeficiency virus (HIV) GP pseudotyped HIV or adeno-associated virus 2 vector entry, indicating that not all virus uptake was enhanced by expression of these molecules. RhoB and RhoC overexpression also significantly enhanced VSV infection. Similarly, overexpression of RhoC led to a significant increase in fusion of EBOV virus-like particles. Finally, ectopic expression of RhoC resulted in increased nonspecific endocytosis of fluorescent dextran and in formation of increased actin stress fibers compared to RhoA-transfected cells, suggesting that RhoC is enhancing macropinocytosis. In total, our studies implicate RhoB and RhoC in enhanced productive entry of some pseudovirions and suggest the involvement of actin-mediated macropinocytosis as a mechanism of uptake of EBOV GP and VSVG pseudotyped viral particles.