*3.4. Safety Profile of Retargeted oHSVs*

Safety of retargeted oHSVs rests on their specificity for cancer cells and on genetic stability and is documented by the following lines of evidence. In vitro, both 1st and 3rd generation retargeted oHSVs infected almost exclusively the cancer cells positive for the targeted receptor and failed to infect or infected very poorly receptor-negative cancer cells and non-cancerous cells, unless they transgenically expressed the targeted receptor [27, 41]. In no cases did the infection of few cells in a culture of receptor-negative cells result

in a virus that could be serially passaged. The viruses exhibit genetic stability in that they have been passaged in cultures for several months (3rd generation) or years (1st generation), without any change in retargeting/detargeting properties. In vivo, upon intratumoral (i.t.) administration, the 1st generation R-115 was detectable only in the tumors, and not in serum or other organs (Figure 3A) [55]. When administered intraperitoneally (i.p.), the 1st generation oHSVs did not cause any pathological signs, including brain infections, even at the highest amounts (2 <sup>×</sup> <sup>10</sup><sup>9</sup> PFU). Under the same conditions, the wt-HSV killed all mice (Figure 3B) [41,56]. In vivo, upon intracranial administration, the 1st generation R-LM113 virus did not infect the brains, whereas the wt-HSV readily did (Figure 3C) [57]. Altogether, the results support the notion that (i) that in vitro infection of human cells only occurs at high level HER2 expression, and (ii) the HER2-retargeted oHSVs do not cause detectable off-target infections in mice. A detailed analysis on bio-distribution to human tissues, especially in tissues with low level HER2 expression, remains to be performed.

**Figure 3.** The retargeted oHSVs are safe in mice upon intraperitoneal, intratumoral or intracranial routes of administration. (**A**) R-115 biodistribution to the indicated organs following four intratumoral injections (1 <sup>×</sup> <sup>10</sup><sup>8</sup> PFU/dose or vehicle), started at d 10 after tumor implantation. Organs and tumors were explanted at d 26, and, after homogenization, the total DNA was extracted. R-115 genome copy numbers were determined by qPCR in comparison with a standard curve obtained with purified HSV DNA, and expressed as gc/100 ng of DNA or gc/100 µl blood. (**B**) Kaplan Meier survival curves of the C57BL/6 mice intraperitoneally injected with 1 <sup>×</sup> <sup>10</sup><sup>8</sup> or 2 <sup>×</sup> <sup>10</sup><sup>9</sup> PFU of R-LM5 (wt HSV), R-LM113 and R-115 (1st generation) oHSVs. (**C**) Merged fluorescence and bright-field images of adult nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse brains after injection with R-LM5 (1 <sup>×</sup> <sup>10</sup><sup>5</sup> PFU) or R-LM113 (3 <sup>×</sup> <sup>10</sup><sup>5</sup> PFU) viruses. Viral spread is visualized by enhanced green fluorescent protein fluorescence. (**A**) Statistical significance was calculated using the Log-rank (Mantel-Cox) test. Panels (**A**–**C**), reproduced with permission. \*\* = *p*-value < 0.01.
