Reduction of nontarget infection and systemic toxicity by targeted delivery of conditionally replicating viruses transported in mesenchymal stem cells

Abstract

The fiber-modified adenoviral vector Delta-24-RGD (D24RGD) offers vast therapeutic potential. Direct injection of D24RGD has been used to successfully target ovarian tumors in mice. However, systemic toxicity, especially in the liver, profoundly limits the efficacy of direct viral vector delivery. Mesenchymal stem cells (MSC) have the ability to function as a vector for targeted gene therapy because of their preferential engraftment into solid tumors and participation in tumor stroma formation. We show that MSC-guided delivery of D24RGD is specific and efficient and reduces the overall systemic toxicity in mice to negligible levels compared with D24RGD alone. In our model, we found efficient targeted delivery of MSC-D24RGD to both breast and ovarian cell lines. Furthermore, immunohistochemical staining for adenoviral hexon protein confirmed negligible levels of systemic toxicity in mice that were administered MSC-D24RGD compared with those that were administered D24RGD. These data suggest that delivery of D24RGD through MSC not only increases the targeted delivery efficiency, but also reduces the systemic exposure of the virus, thereby reducing overall systemic toxicity to the host and ultimately enhancing its value as an anti-tumor therapeutic candidate.

Figure 1. MSC replication

The pattern of MSC growth is a sigmoidal curve. MSC were initially plated at 20,000 cells per well in a six-well plate. After 2 days, MSC exit the lag phase and begin to divide rapidly, about once every 24 hours. This continues through day 7, when the cells are near confluence.

Figure 2. D24RGD efficiently replicates in MSC

MSC were infected with 5–2500 vp/cell of (a) D24RGD or (b) UV D24RGD. A CPE was seen within 48 hours in cells infected with D24RGD but not those infected with UVD24RGD. (c) These data were confirmed by detection of hexon levels in the supernatants of MSC infected with D24RGD or UVD24RGD (5 or 2500 vp/cell). MSC that were infected with D24RGD showed an increase in hexon levels, consistent with amplification of the virus. (d) Burst size data based on E1a expression shows that an MSC infected with an MOI of 50, after 24 hours, was able to produce over 1500 vp per cell.

Figure 3. MSC-D24RGD induces CPE in multiple cell lines

Seventy-two-hour supernatants from MSCs infected with (a) D24RGD or (b) UVD24RGD were applied to multiple cell lines. Evidence of CPE is seen in panel a, confirming that D24RGD can replicate in MSC and produce virus that is able to infect, replicate in, and lyse target cells in vitro.

Figure 4. Viability of tumor cell lines treated with D24RGD-infected MSC supernatant

Supernatants of MSCs conditioned for 48 hours with D24RGD or UVD24RGD were applied to human ovarian carcinoma cell lines OVCAR-3 or SKOV-3. Then, 72 hours later, viable cells were determined via trypan blue counts and displayed as percentage of control counts. The decrease in cell number was inversely proportional to the increase in virus titers used to infect MSC.

Figure 5. Intraperitoneal administration of MSC-D24RGD increases survival of SCID mice

Anti-cancer effect of targeted D24RGD to established Skov-3 ovarian carcinoma via intraperitoneal administration of with and without the MSC vehicle. (a) MSC-D24RGD (4 injections, 1×/week) show a survival benefit (P≤.0001) over that of MSC delivery the UVD24RGD control virus. Survival of SCID mice treated with MSC-D24RGD or D24RGD increased by 55 days on average, compared with survival of control mice. (b) Hexon staining of tumor sections from mice representative of the group receiving D24RGD alone, MSC-D24RGD, and MSC alone treated mice.

Figure 6. Immunohistochemical analysis of mice after D24RGD treatment

Analysis of tissues from SKOV-3 SCID mice 1 or 3 days after intraperitoneal injection of either MSC-D24RGD or D24RGD showed significant differences in the dispersion of hexon. (a) Day 1 after injection of MSC-D24RGD. All tissues except for a small spot in the liver are negative. (b) Day 1 after injection of D24RGD. All tissues except for the lung are positive. The liver is bright with hexon-positive staining. (c) Day 3 after injection of MSC-D24RGD. All tissues again are negative, with the exception of a small spot in the liver. (d) Day 3 after injection of D24RGD. Virus has cleared from muscle tissues, and there is a decrease in liver concentration.

Figure 7. Relative intensity of hexon staining in tissue samples from in vivo analysis

Hexon staining was measured as relative intensity among tissue samples. MSC- D24RGD displayed the least systemic toxicity, which was limited to the liver.