A mouse model study of toxicity and biodistribution of a replication defective adenovirus serotype 5 virus with its genome engineered to contain a decoy hyper binding site to sequester and suppress oncogenic HMGA1 as a new cancer treatment therapy

Abstract

The HGMA1 architectural transcription factor is highly overexpressed in many human cancers. Because HMGA1 is a hub for regulation of many oncogenes, its overexpression in cancer plays a central role in cancer progression and therefore HMGA1 is gaining increasing attention as a target for development of therapeutic approaches to suppress either its expression or action in cancer cells. We have developed the strategy of introducing decoy hyper binding sites for HMGA1 into the nucleus of cancer cells with the goal of competetively sequestering overexpressed HMGA1 and thus suppressing its oncogenic action. Towards achieving this goal, we have introduced an HMGA1 decoy hyper binding site composed of six copies of a high affinity HMGA1 binding site into the genome of the replication defective adenovirus serotype 5 genome and shown that the engineered virus effectively reduces the viability of human pancreatic and cancer cells. Here we report the first pre-clinical measures of toxicity and biodistribution of the engineered virus in C57BL/6J Black 6 mice. The immune response to exposure of the engineered virus was determined by assaying the serum levels of key cytokines, IL-6 and TNF-α. Toxicity due to exposure to the virus was determined by measuring the serum levels of the liver enzymes aspartate aminotransferase and alanine aminotransferase. Biodistribution was measured following direct injection into the pancreas or liver by quantifying viral loads in the pancreas, liver, spleen and brain.

Fig 1. Change of body weight after direct injection of virus into the pancreas or liver.

(A) Change of mice body weight after injecting the AdEasy or AdEasy-HMGA-6 virus into the liver. (B) Change of mice body weight after injecting the AdEasy or AdEasy-HMGA-6 virus into the pancreas. Mice were injected with 1.0X10⁸ virus particles/kg of body weight. Data are shown for the 30-day time-period group. Mouse body weight was measured before injection of the viruses or 20 μL PBS (as vehicle control) and monitored until euthanization at indicated time points. The body weight change (on vertical axis) indicates the difference between the weight before injection and the weight at indicated time points. All data represent the means ± SD of three mice.

Fig 2. Determination of virus copy number after injecting virus into the pancreas or liver.

(A) Viral copy number in different organs after injecting AdEasy into the liver. (B) Viral copy number in different organs after injecting AdEasy-HMGA-6 into the liver. (C) Viral copy number in different organs after injecting AdEasy into the pancreas. (D) Viral copy number in different organs after injecting AdEasy-HMGA-6 into the pancreas. Mice were injected with 1.0X10⁸ virus particles/kg of body weight. Viral copy numbers were determined by qRT PCR using five microliters of total genomic DNA isolated from each tissue as template. The AdEasy and AdEasy-HMGA-6 groups had n = 12 and liver, pancreas, spleen and brain were collected 6h, 3, 7, 10 and 30 days post-injection. Tissues from the vehicle control group (n = 3) were collected after 3 days. The sham control group (n = 3) did not undergo any surgical procedure. Quantification cycle (Cq) values less than the negative control were considered negative. All data represent the means ± SD of three mice.

Fig 3. Determination of serum TNFα and IL-6 following injection with virus.

(A) Serum levels of TNFα following virus injection into liver. (B) Serum levels of TNFα following virus injection into pancreas. (C) Serum levels of IL-6 following virus injection into liver. (D) Serum levels of IL-6 following virus injection into pancreas. Serum levels of TNFα or IL-6 were determined by ELISA following injection 1.0X10⁸ virus particles / kg of body weight or PBS (20μL) as vehicle control. Serum samples were collected 6h, 3, 7 or 30 days post-injection of viruses or PBS. The sham control group did not undergo any surgical procedure. All data represent the means ± SD of three mice.

Fig 4. Analysis of serum transaminases following injection with viruses.

(A) ALT concentration following injection with AdEasy, AdEasy-HMGA-6 or PBS as vehicle control into the liver. (B) ALT concentration following injection with AdEasy, AdEasy-HMGA-6 or PBS as vehicle control into the pancreas. (C) AST concentration following injection with AdEasy, AdEasy-HMGA-6 or PBS as vehicle control into the liver. (D) AST concentration following injection with AdEasy, AdEasy-HMGA-6 or PBS as vehicle control into the pancreas. Viruses were injected with a dose of 1.0X10⁸ virus particles / kg of body weight. 20μL of PBS was injected as a vehicle control. The sham control group did not undergo any surgical procedure. Serum samples were collected 6h, 3, 7 or 30 days post-injection of viruses or PBS. All data represent the means ± SD of three mice.