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The skin is an attractive tissue for gene therapy applications to treat genetic disorders and to express systemically delivered transgenes encoding therapeutic proteins. Understanding the tissue tropism of vectors is a prerequisite for the design of gene therapy trials. Using an ex vivo system of organ culture, we studied factors that determined viral tropism to the epidermal and dermal cells in human and mouse skin. We applied in these studies a lentiviral vector pseudotyped with two glycoproteins that use different cell receptors (vesicular stomatitis virus glycoprotein [VSV-G] and amphotropic murine leukemia virus envelope). The extent of infection with the amphotropic pseudotype was much higher than that of VSV-G, especially at low multiplicities of infection. In contrast, the tropism of these two pseudotypes in skin tissues was similar; at low multiplicities the infection was limited to areas near the basal layer of the epidermis, whereas at high multiplicities the infection extended to the dermal layer. To overcome physical barriers in the skin, the epidermal and dermal layers were separated and infected. Whereas the human epidermis was readily infected, we could not detect infection of stem and early progenitor cells in their niche. In contrast, mouse epidermis was completely resistant to infection. Dermal cells of both species were readily infected with the two pseudotypes. Molecular analysis indicated that infection of mouse epidermal cells was restricted after proviral DNA synthesis and before integration. In conclusion, we show that lentiviral tropism in a solid tissue is dependent on several factors, extra- and intracellular, distinct of the cellular receptors.
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Although more adeno-associated virus AAV-based drugs enter the clinic, vector tissue tropism remains an unresolved challenge that limits its full potential despite that the tissue tropism of naturally occurring AAV serotypes can be altered by genetic engineering capsid vie DNA shuffling, or molecular evolution. To further expand the tropism and thus potential applications of AAV vectors, we utilized an alternative approach that employs chemical modifications to covalently link small molecules to reactive exposed Lysine residues of AAV capsids. We demonstrated that AAV9 capsid modified with N-ethyl Maleimide (NEM) increased its tropism more towards murine bone marrow (osteoblast lineage) while decreased transduction of liver tissue compared to the unmodified capsid. In the bone marrow, AAV9-NEM transduced Cd31, Cd34, and Cd90 expressing cells at a higher percentage than unmodified AAV9. Moreover, AAV9-NEM localized strongly in vivo to cells lining the calcified trabecular bone and transduced primary murine osteoblasts in culture, while WT AAV9 transduced undifferentiated bone marrow stromal cells as well as osteoblasts. Our approach could provide a promising platform for expanding clinical AAV development to treat bone pathologies such as cancer and osteoporosis. Thus, chemical engineering the AAV capsid holds great potential for development of future generations of AAV vectors.
Recombinant adeno-associated virus (rAAV) gene therapy has a broad clinical impact on improving patient outcomes for various genetic diseases, including the three FDA-approved AAV-drugs: Luxturna (treatment for Leber congenital amaurosis), Zolgensma (treatment for spinal muscular atrophy), and recently Hemgenix (treatment for Hemophilia B)1. More than one hundred clinical trials using AAV drugs have been conducted to correct human defective genes, making AAV the most popular gene delivery system, mostly due to their relatively low immunogenicity, customizable design, and safety profile. Despite the fast pace of success for AAV vectors, unresolved challenges still exist such as limited tropism, poor transduction efficacy in certain tissues, pre-existing neutralizing antibodies (NAbs), and high vector dosage2.
Here, our study utilized a chemical modification approach to covalently link small molecules to reactive exposed Lysine residues of capsids AAV2, AAV8 and AAV9. From in vitro transduction screening, AAV9 capsid modified with N-ethyl Maleimide (NEM) was shown to best enhanced the gene expression of in human endothelial cell line, therefore NEM was chosen for further in vivo analyses in this study. Herein, we demonstrate that AAV9-NEM increased the transduction of bone marrow while decreased transduction of liver tissue in vivo, compared to unmodified WT AAV9, and that chemical engineering the AAV capsid with small molecules could bring great potential for future generation of AAV vectors.
As shown in Fig. 3B, DNA analysis of tissues at week 1 show the early changes in tropism of AAV9-NEM vs. AAV9, as that AAV9-NEM moderately increased vector DNA in some tissues (e.g., liver, kidney, or spleen) while decreased in others (e.g., bone marrow, lungs, or stomach). At week-4 post-injection, the change in tropism of AAV9-NEM was found to be more enhanced in the bone marrow, where vector genome (vg) DNA was around sevenfold higher than the unmodified AAV9 (Fig. 3C). The absolute vg detected corresponding to Fig. 3B is reported in Fig. S1. To examine the gene expression (gLUC), in vivo bioluminescence imaging was used with IP injection of the substrate coelenterazine with a dose of 200 mL/mouse (30 mg/mouse) and performed immediately after the substrate injection. AAV9-NEM gave a brighter signal (double the light intensity emitted) compared to AAV9 at week-4 post injection (Supplemental Fig. S2). These results indicate that the chemical-engineered capsid AAV9-NEM indeed changed the tropism and also enhanced the transduction of wild-type capsid AAV9.
To further investigate observed differences between AAV9 and AAV9-NEM detected in the livers and bone marrow, we designed experiments using fluorescent proteins to compare transduction of specific regions of these two organs. We chose AAV9-eGFP (packaged pdsAAV-CB-eGFP) and AAV9-mScarlet (packaged pdsAAV-CB-mScarlet) modified with NEM for the in vivo study, as well as lengthened the time course to better understand the dynamics of NEM modification of AAV917. At 4- and 8-weeks post injection, soft organs and hindlimbs were collected and processed for histological, fluorescent, and flow cytometry analyses. Liver sections revealed a slight decrease in AAV9-NEM positive cells, indicated by mScarlet expression, compared to AAV9-eGFP transduced cells (Fig. 4). This trend matches the vg content data presented in Fig. 3. At 8 weeks, both eGFP and mScarlet positive cell numbers decreased, yet at both timepoints, there were only few double positive eGFP and mScarlet liver cells. When liver sections were stained for the vascular marker Endomucin (Emcn) and the Kupffer cell/macrophage marker F4/80, there was minimal overlay between these antibodies and both populations of AAV9-transduced cells compared to when sections were stained with hepatocyte marker Albumin; this was observed at 4 weeks and 8 weeks post injection18,19. This data suggests that most of the cells being transduced with AAV9 in the liver are most likely hepatocytes and not endothelium or phagocytic cells present in the tissue20,21,22.
In the bone marrow, we observed a 4% and a 2.5% difference between AAV9-eGFP and AAV9-NEM-mScarlet via flow cytometry and immunofluorescence, respectively, at 4 weeks post injection (Fig. 5A, B). As we observed in the liver, the number of double positive cells in the bone marrow was significantly lower compared to the single-positive groups. We observed a similar difference in bone marrow at 4 weeks when the fluorescent transgenes were exchanged between the AAV9 vectors (Supplemental Fig. S3). By 8 weeks, all populations in the bone marrow decreased to below 2% using both methods of analysis. Of note, fluorescent imaging showed distinct areas of green and red positivity in the hindlimbs; the marrow component contained strong eGFP green signal, and a prominent mScarlet red signal was observed at the interface between the calcified tissue and the marrow tissue, suggesting distinct populations of bone cells are being transduced with the AAV9-NEM vs. AAV9 (Fig. 5C). Indeed, flow cytometric analysis shows that AAV9-NEM-mScarlet cells exhibited a higher % of single-positive cells with vascular markers Cd31 and Cd34, and markers of MSCs with osteogenic potential Cd90, Cd105, and Cxcr4 at 4 weeks compared to AAV9-eGFP cells23; Cd31 and Cd90 differences persisted at the 8-week timepoint (Supplemental Fig. S4).
Given that Cd31, Cd34, and Cd90 are markers for vascular cells and to certain extent immune cells, we stained the bone sections with Emcn and F4/80 to investigate more detail about which cell types are being transduced24,25. Interestingly, Emcn overlayed strongly with mScarlet and less so with eGFP in our analysis at both 4 and 8 weeks. This pattern was not observed with F4/80; there was minimal co-localization with either eGFP or mScarlet populations (Fig. 5D). Overall, these results suggest that AAV9 transduction pattern is different between the liver and the bone marrow, and that the NEM modification further alters that pattern of AAV9 transduction.
The location of the mScarlet-positive AAV9-NEM cells in the bone tissues of the mice, and the presence of Cd90 and Cxcr4-positive AAV9-NEM bone marrow suggest that cells involved in bone turnover could be a target of this modified AAV9 vector26. To investigate these potential transduction differences between AAV9 and AAV9-NEM in a more controlled environment, we isolated primary mouse bone marrow stromal cells (BMSCs) from bone marrow of uninjected Balb/C male adult mice for two sets of studies. We employed AAV9 or AAV9-NEM driving expression of gLUC under control of the CB promoter for the first set of experiments, and AAV9-CB-eGFP along with AAV9-NEM-CB-mScarlet for the second set. At Days 3 and 5 of the gLUC experiments, BMSCs transduced with AAV9 (MOI of 104) exhibited significantly higher luciferase readout compared to AAV9-NEM. Interestingly, BMSCs transduced on Day 8 (48 h in osteogenic medium) with either AAV9 or AAV9-NEM resulted in similar luciferase readings. This trend continued to be seen on Days 14 and 21 (Fig. 6A). This data suggests that the NEM modification of AAV9 alters its transduction in vitro. 152ee80cbc
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