br the human PDAC tumors
1. the human PDAC tumors were implanted subcutaneously, as opposed to orthotopic tumors; 2. human PDAC (±)-Baclofen selected for the study ex-press BIRC5, whereas not all PDAC tumors overexpress BIRC5; 3. BIRC5 is expressed in low levels benign human tissues.
Tumor promoter-driven reporter assays for detection of gene ex-pression have been used previously. However, endogenous promoters are unable to detect low levels of gene expression. For this reason, we chose to utilize an enhanced super-promoter of the PDAC overexpressed gene, BIRC5. The BIRC5-SPTSTA resulted in greater than 100-fold in-
crease of promoter activity compared to endogenous BIRC5 promoter, thus it is capable of detecting low levels of BIRC5 expression in both engineered cells and PDAC cells. While synthetic promoters and the TSTA system have been studied separately , the strategy of com-bining them into an enhanced BIRC5 super-promoter for exponential reporter gene expression is novel. The limitation of using the enhanced BIRC5 super-promoter is that it could detect low levels of BIRC5 ex-pressed in benign tissues of the human body and thus lead to false-positives. However, the magnitude of such background levels would be well-defined upon continued studies of this platform.
GLuc and sr39TK reported genes were selected for the diagnostic platform, allowing simultaneous serologic testing, optical imaging and microPET/CT imaging. While it is known that GLuc is secreted from PDAC cells and can be readily measured in blood over time, rising serologic GLuc levels correlated with increasing PDAC tumor volume, demonstrating remarkable sensitivity of serologic GLuc for detection of minute PDAC in mice . The diﬀerence in blood volume between mice and humans represents a potential limitation of detection of ser-ologic GLuc. Optical imaging is not available for clinical uses. There-fore, a PET/CT reporter gene was added to the vector for future translational purposes, as pre-clinical and clinical trials have demon-strated the feasibility of sr39TK in PET imaging of tumors . Using GLuc-2A-sr39TK as a dual expression vector, we demonstrated that GLuc and sr39TK were well-cleaved without influencing their activity . Quantification of [18F]FHBG imaging in our study demonstrated that sr39TK localized minute human PDAC tumors bearing GLuc-2A-sr39TK in mice, therefore GLuc-2A-sr39TK serves as a single vector for simultaneous serologic detection and PET/CT localization. [18F]FHBG is an ideal substrate for the HSV1-sr39TK . The highly specific PDAC tumor images in this feasibility study with no visible background noise, including in the pancreas and GI tract after a clearance period of three hours, makes it suitable for imaging HSV1-sr39TK gene expres-sion .
We chose AAV2 as the delivery system, as its safety profile has long been established and it is the only FDA approved gene therapy vector available to date [16,17,39,40]. Due to non-specific limitations of AAV2, alterations of AAV2 tropism for cancer is an important aspect of the strategy for targeted delivery of the reporter genes [41–43]. Studies
have shown that replacement of heparan sulfate proteoglycan (HSPG) on the viral capsid with high-aﬃnity peptides permits the virus to bind specifically to tumors and prevents binding to normal cells [44,45]. Münch et al. inserted ankyrin repeat proteins specific to the HER2 re-ceptor at the N terminus of the VP2 region of the AAV2 capsid, thereby increasing the specificity of the vector to tumor cells overexpressing the HER2 receptor by ∼30-fold in vitro and ∼20-fold in vivo . We ge-netically engineered the AAV2 capsid with incorporation of a panel of PDAC-targeting tumor homing peptides at site R588, which have been validated to have high binding aﬃnity to the surface membrane of tumors, thus improving viral infectivity and specificity [42,43,46]. AAV2RGD showed the highest infection of PDAC cells due to high ex-pression of integrin αvβ3, which is consistent with other reports [44,47]. However, non-specific background signals have been observed when AAV2RGD is utilized alone due to expression of avb3 in benign
tissues, therefore, AAV2RGD was coupled with the enhanced BIRC5-SPTSTA to drive GLuc-2A-sr39TK dual reporter genes specifically in
PDAC, as seen in Fig. 1. Conversely, the use of the wildtype AAV2 or the engineered virus with a CMV promoter demonstrated non-specific leakage of reporter signals in benign mouse tissues. Therefore, the combination of the engineered AAV2 and an enhanced BIRC5 super-promoter resulted in strong reporter signals only in minute human PDAC tumors with no leakage.
We recognize the future risk of administering an engineered AAV2 to patients and thus envision that the platform would be used for pa-tients who are undergoing high risk surgery for pancreatic cancer. Therefore, the rationale for developing the platform is to diﬀerentiate PDAC from currently undetectable metastases or benign pancreatic masses to improve surgical outcomes for patients with resectable PDAC and to avoid unnecessary surgery. We anticipate that the diagnostic platform would be used for patients who are about to undergo surgery for resectable masses of the pancreas. For example, the vector would be given one week before surgery, then the patient would undergo PET/CT imaging and serologic testing for Gaussia luciferase pre-operatively to determine whether the primary tumor is malignant or benign and whether there are metastases. If the tumor is benign or if there is me-tastatic disease, surgery would be avoided. One month post-operatively, serologic testing for Gaussia luciferase and PET/CT would be performed to determine whether all PDAC has been resected or whether any re-sidual tumor or metastases are present. If the platform can reduce un-necessary surgeries by determining whether there is metastatic PDAC or benign pancreatic masses, the risk of the viral vector would be justifi-able. Patients with truly resectable PDAC would experience a marked improvement in survival following surgery. The data from this feasi-bility study support the rationale to proceed with further studies de-signed for translation of the precision diagnostic platform for detecting and localizing minute human PDAC.