Adenoviral-mediated imaging of gene transfer using a somatostatin receptor-cytosine deaminase fusion protein

Abstract

Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzyme’s ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy.

A brief review of reporter gene imaging in oncolytic virotherapy and gene therapy

“… 95 SSTR2 has been inserted into adenovirus and oncolytic vaccinia, where long-term tracking of viral infection and persistence was shown to be feasible in several tumor types. 32 , 45 , 96 , 97 , 98 , 99 SSTR2 has also been used to track AAV gene therapy, where it permitted PET imaging 6 months post-treatment. 100 The major drawbacks of SSTR2 are endogenous expression, which can reduce diagnostic efficacy, and that each receptor can only bind one radiolabeled ligand, preventing signal amplification and thus limiting imaging sensitivity.…”

Expanding Theranostic Radiopharmaceuticals for Tumor Diagnosis and Therapy

“…However, the GEN2 directed cancer immunotherapy trial is the only one that includes monitoring of HSV-1-tk-m2 expression using [ 18 F]FHBG PET imaging (NCT04313868) while its use to monitor HSV-1-tk gene therapy in humans has already been validated [105], including the use of [ 124 I]FIAU as the reporter probe [92] The human somatostatin receptor has also been used as a preclinical platform for imaging reporter gene therapy [106]. The human somatostatin receptor subtype 2 (hSSTR2) has been used as (i) reporter gene for adenoviral (AdV)-mediated gene transduction into preclinical non-small cell lung cancer [107] and breast cancer models [108] and visualized using [ 68 Ga]Ga-DOTATATE-and [ 68 Ga]Ga-DOTATOC PET imaging [109,110]; (ii) in a theranostics paradigm employing [ 90 Y]Y-DOTATOC in hSSTR2-transduced xenografts [111]; (iii) in oncolytic virus (OV) therapy approaches [112,113]; and (iv) to study the dynamics of CAR T-cell responses in a mouse model of thyroid cancer [114].…”

Discovery and directed evolution of nitroreductase enzymes for activation of prodrugs and PET imaging compounds

“…There has been less success creating a CD-imageable reporter probe, with attempts at utilizing 3 H-labelled 5-FC for both gene therapy and PET imaging showing no difference in uptake of 5-FC between a human glioblastoma cell line stably expressing bacterial CD, and control cells (Haberkorn et al, 1996). Some imaging success has been achieved by indirect measurement of CD localisation through intratumoural injection of a somatostatin receptor subtype 2 (SSTR2)-CD fusion protein in a replication-deficient adenovirus to mice xenograft tumours (Lears et al, 2015). Administration of the PET imaging probe 64 C-CB-TE2A-Y3-TATE showed activation to a cell-entrapped form by SSTR2 in mice administered the adenovirus containing the fusion protein, and activation of 3 H-5-FC by the CD enzyme could also be detected, showing that both parts of the fusion protein were still active (Lears et al, 2015).…”

“…Some imaging success has been achieved by indirect measurement of CD localisation through intratumoural injection of a somatostatin receptor subtype 2 (SSTR2)-CD fusion protein in a replication-deficient adenovirus to mice xenograft tumours (Lears et al, 2015). Administration of the PET imaging probe 64 C-CB-TE2A-Y3-TATE showed activation to a cell-entrapped form by SSTR2 in mice administered the adenovirus containing the fusion protein, and activation of 3 H-5-FC by the CD enzyme could also be detected, showing that both parts of the fusion protein were still active (Lears et al, 2015).…”