Phase 1 Trial of Bi-shRNA STMN1 BIV in Refractory Cancer

Abstract

Stathmin1 (STMN1) is a microtubule modulator that is expressed in multiple cancers and correlates with poor survival. We previously demonstrated in vivo safety of bifunctional (bi) shRNA STMN1 bilamellar invaginated vesicle (BIV) and that systemic delivery correlated with antitumor activity. Patients with superficial advanced refractory cancer with no other standard options were entered into trial. Study design involved dose escalation (four patients/cohort) using a modified Fibonacci schema starting at 0.7 mg DNA administered via single intratumoral injection. Biopsy at baseline, 24/48 hours and resection 8 days after injection provided tissue for determination of cleavage product using next-generation sequencing (NGS) and reverse transcription quantitative polymerase chain reaction (RT-qPCR), 5' RLM rapid amplification of cDNA ends (RACE) assay. Serum pharmacokinetics of circulating plasmid was done. Twelve patients were entered into three dose levels (0.7, 1.4, 7.0 mg DNA). No ≥ grade 3 toxic effects to drug were observed. Maximum circulating plasmid was detected at 30 seconds with less than 10% detectable in all subjects at 24 hours. No toxic effects were observed. Predicted cleavage product was detected by both NGS (n = 7/7 patients analyzed, cohorts 1, 2) and RLM RACE (n = 1/1 patients analyzed cohort 3). In conclusion, bi-shRNA STMN1 BIV is well tolerated and detection of mRNA target sequence-specific cleavage product confirmed bi-shRNA BIV mechanism of action.

Figures

Figure 1

Whole blood pharmacokinetics of bi-shRNA STMN1. Blood was collected at a distal location away from the intratumoral injection site at defined time points. Hundred microliters of whole blood were extracted for DNA. Hundred nanograms of each sample were analyzed by quantitative polymerase chain reaction for the presence of bi-shRNA STMN1. The detected plasmid was normalized to the total DNA extracted per 100 µl of blood sample. Panel (a) shows the highest copies of plasmids detected from each patient's time course samples. Panel (b) shows a typical pharmacokinetics within 1 hour postinjection (patient #1007). Panel (c) shows the pharmacokinetics of cohort 2 patient samples between 1 hour and 24 hours postinjection.

Figure 2

Demonstrate mechanism of drug action: detection and identification of RNAi-mediated target mRNA cleavage product. RNA ligase-mediated RACE (RLM-RACE) and next-generation sequencing (NGS) were employed to identify RNAi-mediated target mRNA cleavage in human tumors with intratumoral injections of bi-shRNA STMN1 BIV. Total RNA were extracted from needle biopsies of patient tumor and analyzed for the presence of RNAi mediated cleavage product. (a) Sequence alignment of NGS data on treated tumor samples from cohort 1 patients and HCT-116 cells treated with bi-shRNA STMN1 for positive control (right panel), and from cohort 2 patients (left panel). Red rectangular boxes indicate the correct cleavage product. (b) A 4% agarose gel image of RLM-RACE analysis on tumor biopsy samples from patient #1013. Lane L is 100 bp DNA size marker, lane 1 is HCT-116 cells treated with bi-shRNA STMN1, lane 2 is HCT-116 cells without treatment, lane 3 is patient #1013 pretreatment sample, lane 4 is patient #1013 24 hours post-treatment biopsy, lane 5 is patient #1013 day 7 post-treatment sample. Arrow indicates the correct 269 bp cleavage product band, the correct size band was sequence confirmed. (c) A representative sequence chromatograph of the STMN1 cleavage product. The broken arrow indicates the RNA adaptor which was used to ligate the cleavage product. The filled arrow indicates human STMN1 sequence. A diagram illustrates the RNAi-mediated cleavage site of human STMN1 mRNA.

Figure 3

Target gene knockdown assessment by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Normalized STMN1 mRNA level with RNA isolated from biopsy samples of patients 1002, 1006, 1011, and 1013. RT-qPCR was normalized to GAPDH by ▵▵Ct method. Triplicates were performed for each RT-qPCR and compared to STMN1 mRNA level in HCT-116 cells. Each sample is designated by patient number and biopsy harvest time. BL, base line; D3, day 3 postinjection; D2, day 2 postinjection; D7, day 7 postinjection; D8, day 8 postinjection. GAPDH, glyceraldehyde-3-phosphate dehydrogenase