The tumoricidal activity of the HSV-TK/GCV system is due to several factors. In dividing cells, the 
phosphorylated GCV inhibits DNA synthesis. This effect is not confined to cells that are directly 
transduced with HSV-TK, as neighboring cells are also affected. This phenomenon, which likely occurs 
as a result of several mechanisms, has been termed the “bystander effect" and has been observed in 
several tumor types including CNS tumors. Transfer of the phosphorylated GCV between cells, 
(“metabolic cooperation') via gap juctions has been proposed as a possible mechanism. Phagocytosis by 
neighboring cells of GCV phosphate containing apopotic vesicles (from dying transduced cells) has also 
been proposed [33], Immune-mediated processes may also account for significant killing of non- 
transduced cells. In one report [28], anti-tumor immunity was observed following TK mediated killing 
of experimental brain tumors. This protective immunity led to eradication of subsequent tumor cell 
inoculi when the animal was rechallenged at a remote site [28]. Whether the tumor immunity is TK 
dependent, or merely a manifestation of inherent tumor cell immunogenicity, has yet to be establihed in 
this rodent model. 
These important findings have served to point out the limitation of this approach. Golumbek et. al. [25] 
demonstrated that HSV-TK was unable to completely eliminate non-immunogeniec tumor cells even 
when 100% of the introduced cells exhibited TK expression. They observed a delayed outgrowth of TK + 
tumor cells occurring many days after cessation of GCV. These cells were likely in Go arrest during the 
interval of GCV therapy and thus were able to escape the toxic effects of GCV metabolites. Alternatively, 
they were sequestered in an area of poor GCV penetration and thus received sublethal doses of the drug. 
II. B. 2 Toxicity of Ganciclovir 
Ganciclovir is a synthetic nucleoside analog which is approved for the treatment of CMV retinitis in 
patients with acquired immunodeficiency syndrome and for the prevention of CMV infection in bone 
marrow transplant recipients. In addition, it is used to treat CMV infections in transplant patients. 
Extensive clinical experience exists in the use of this drug. It is distrubes widely to all tissues 
including the CSF and brain parenchyma (see Appendix VI.A, Page 24). Ganciclovir has been shown to 
exhibit several toxic side effects in humans. Mammalian cell proliferation (in particular bone marrow) 
is affected (in vitro) at high concentrations (10 to 60 mg/ml). Granulocytopenia and thrombocytopenia 
have been observed in patients treated with ganciclovir. Renal impairment has been attributed to 
ganciclovir, especially when used in combination with other nephrotoxic drugs. Seizures have been 
observed when this drug was administered in combination with imipenem-cilastatin. It has been shown 
to be carcinogenic, teratogenic and caused aspermatogenesis in animal studies. Headache and confusion 
have been observed in transplant patients at frequencies of 17% and 6%, respectively. Other frequent 
adverse effects include anemia, fever, rash and abnormal liver function tests. Each of these occurred in 
approximately 2% of patients. 
II. B. 3 The Clinical Spectrum of Wild-Type Adenovirus Infections of the CNS 
Adenovirus CNS infection is sporatic and uncommon, although its true incidence can not be estimated 
from case reports. Undoubtedly many cases are neither diagnosed (aseptic meningitis) nor reported. 
From the reports available adenovirus meningoencephalitis is usually self-limited and the clinical 
outcomes governed by the the presence of underlying disease in the affected patients. We anticipate that 
the use of a replication defective adenovirus will substantially limit the spead of the virus and attentuate 
the pathologic consequences. This will be established more definatively in our ongoing toxicity studies. 
A brief summary of clinical reports of wild-type adenovirus infections of the CNS is provided in 
Appendix VI. E, page 33. 
II. B. 4 Experimental Wild-Type Adenovirus Infections of the CNS In Primates. 
In the course of developing an adenoviral vaccine the CNS toxicity of wild-type adenovirus strains were 
studied. Rorke and colleagues [34, 35] systematically studied the neurovirulence of several adenovirus 
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