KRAS mutant allele-specific expression knockdown in pancreatic cancer model with systemically delivered bi-shRNA KRAS lipoplex

The KRAS oncogene, present in over 90% of pancreatic ductal adenocarcinomas, is most frequently the result of one of three gain-of-function substitution mutations of codon 12 glycine. Thus far, RAS mutations have been clinically refractory to both direct and selective inhibition by systemic therapeutics. This report presents the results of pre-clinical assessment of a lipoplex comprising a plasmid-encoded, modular bi-functional shRNA (bi-shRNA), which executes selective and multi-mutant allelic KRASG12mut gene silencing, encased within a fusogenic liposome systemic delivery vehicle. Using both a dual luciferase reporter system and a Restriction Fragment Length Polymorphism (RFLP) assay, selective discrimination of KRASG12mut from KRASwt was confirmed in vitro in PANC1 cells. Subsequently, systemic administration of the bi-shRNAKRAS fusogenic lipoplex into female athymic Nu/Nu mice bearing PANC1 xenografts demonstrated intratumoral plasmid delivery, KRASG12mut knockdown, and inhibition of tumor growth, without adverse effect. Clinical trials with the bi-shRNA lipoplex have been implemented.


Background
Pancreatic ductal adenocarcinoma (PDAC) is a disease characterized by early metastatic spread and high mortality. There has been limited benefit from the incremental changes in therapy of PDAC over the past 40 years despite an increased understanding of the genetic, epigenetic, biochemical and micro-environmental processes of this malignancy [1][2][3][4]. Mutations involving the proto-oncogene KRAS and tumor suppressors CDKN2A, TP53 and SMAD4 are the major genetic signal alterations responsible for malignant phenotype [2,5,6]. More than 90% of PDAC's contain KRAS activating mutations, the majority of which are at codon G12 (COSMIC database). These mutated RAS family genes are key "pro-cancer" regulators of the RAF/MEK/ERK, PI3K/AKT/mTOR and RalA/B signaling pathways [4,7]. Recently, in vitro and in vivo targeting of MEK, ERK, PI3K and mTOR in pancreatic cancer have shown promising results based on their ability to impede cellular growth or delay tumor formation. Several clinical trials have been initiated based on these results (NCI-2016-01356) [2,[8][9][10]

Cell viability assay
Cell viability was assayed using the CellTiter-Blue1 Luminescence Cell Viability Assay System from Promega (Madison, WI). Transfected cells or treated cells were plated in triplicate in 96-well plates and assayed at 24, 48 or 72 hours post transfection. Cells were lysed and assayed with reagents supplied by the assay system and the fluorescence was detected by using Lumi-noskan™ Ascent Microplate Luminometer (ThermoFisher Scientific).

DNA and DNA-Lipoplex
50 mg of research grade plasmid DNA was contract manufactured by Aldevron (Fargo, ND). The identity of manufactured plasmids was reconfirmed by restriction digest and by sequencing the insert region before DNA-lipoplex manufacturing. The lyophilized DNA-Lipoplex was manufactured according to the thin film Liposome method as previously published (Templeton, Nature Biotech 1997 and Phadke, DNA and Cell Biol 2011) with the following modifications: after the rotovap step to create the DOTAP:Cholesterol film, the product was resuspended in 10% sucrose and then manually extruded through successively smaller pore size filters to create the Liposomes. The Liposomes were mixed with DNA to create the DNA-Lipoplex product and intermediate QC was performed to check specifications. The product was vialed, frozen, and lyophilized overnight. The following day, the freeze-dried product was sealed, labeled, and quarantined for QC/release. After the product was released and ready for use, the freeze-dried DNA-Lipoplex was reconstituted in 5% dextrose and extruded through a 1.0 μm filter. At this point, the product was ready for use and could be directly injected or diluted with additional 5% dextrose to the appropriate concentration prior to administration. improved detection sensitivity. 1μl of digested amplicons was loaded onto a DNA 1K chip and the fragments were visualized and analyzed by the Experion analysis software.

Plasmid detection and quantification
The mouse tissues were thoroughly homogenized using Qiagen TissueLyzer II. Tissue homogenate was then digested with proteinase K and the total DNA extracted using DNeasy Kit (Qiagen). The plasmid was detected and quantified using a home-developed qPCR assay. Briefly, 2ul of extracted total DNA was mixed with a BioRad IQ Supermix, a pUMVC3 forward primer, a pUMVC3 reverse prime and a TaqMan probe specifically recognizing the pUMVC3 amplicon. The 40 cycle qPCR program (using a BioRad CFX384 qPCR instrument) was set up using an automatic liquid handler. The DNA copy number was quantified by referring the Ct numbers to a standard curve.

In vivo mouse xenograft study
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Altogen Labs, Austin, TX (IACUC protocol 3-17836). Following modifications were made to the study "endpoint" definition: Moribund animals or tumor xenograft volumes of 2,000 mm 3 , or 40 days after xenotransplantation. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Immune-compromised nude mice (9-to 11-week old females) were purchased from the Harlan laboratories. All animal procedures and maintenance were conducted in accordance with the institutional guidelines (Altogen Labs, Austin, TX). The maximum tumor size was 2,000 mm 3 . Animals were observed at 6-8 hours and 1 day after each injection for acute reaction; neither adverse reactions nor aberrant behavioral phenotypes were observed.

Observation and data collection
After tumor cells inoculation, the animals are checked daily for morbidity and mortality. At the time of routine monitoring, the animals are checked for any adverse effects of tumor growth and treatments on normal behavior such as mobility, visual estimation of food and water consumption, body weight gain/loss, eye/hair matting, pain/distress, self mutilation, and any other abnormal effects. Signs of graft rejection, infection, and unalleviated pain will be justification for immediate euthanasia as determined by the veterinarian.
Tumor volumes are measured every 3-4 days in two dimensions using an electronic caliper, and the volume data are expressed in mm 3 using the formula: V = 0.5 a x b 2 where a and b are the long and short diameters of the tumor, respectively. Dosing and tumor volume measurement procedures are conducted in a Laminar Flow Cabinet according to Altogen Labs IACUC regulations.

Group assignment
Before grouping and treatment, all animals are weighed and the tumor volumes confirmed (100-150mm 3 ) using electronic caliper. Since the tumor volume can affect the effectiveness of any given treatment, mice assigned into groups using randomized block design as following: First, the experimental animals are divided into homogeneous blocks based on their tumor volume. Secondly, within each block, randomization of experimental animals to different groups conducted. By using randomized block design to assign experimental animals, we ensure that each animal has the same probability of being assigned to any given treatment groups and therefore systematic error is minimized.

Clinical observations
There was no clinical signs or behavioral phenotype observed within the study (daily cage intensive observation for adverse effect were performed). No BWL>20% were observed in any of the groups (Fig 1).
Tumor size was measured every 4 days. Animal body weight (g) was measured in subcutaneous PANC1 xenografts on days 7, 14, 21, 28, 35, 39 after tumor inoculation (Day 0); no significant changes were observed. At the end of study, animals were sacrificed by cervical dislocation. The pancreas carcinoma PANC1 (CRL-1469) cell line was obtained from ATCC and cultured in ATCC formulate Dulbecco's Modified Eagle's Medium (cat#302002) and supplemented with fetal bovine serum to a final concentration of 10% (ATCC). Subculturing was performed by trypsinization with 0.25% Trypsin-EDTA (2-3 minutes) and 1:4 split for every subsequent passage. The cell line was cultured at 37˚C / 5% CO 2 in a humidified incubator. Cells were mixed (1:1 volume) with Matrigel (BD Biosciences) and the suspension (50% matrigel) subcutaneously injected (1.0 x 10 6 cells per injection) on day 0 into the animal flank area to ensure successful tumor initiation and tumor growth measurements. Ninety animals were used for PANC-1 xenotransplantation and 78 animals with measurable tumors were selected  on day 7 to be used for subsequent experiments. 10 animals per group (n = 6) were used for growth inhibition study and 3 animals per group (n = 6) were used for molecular analysis.
Freeze-dried formulations used for this study were reconstituted immediately prior to each injection. Reconstitution was performed in a Biological Safety Cabinet. A 3 mL syringe and 16 G needle was used to transfer D5W into the vial containing the freeze-dried test article, which was then gently flicked to resuspend the test article (final concentration = 0.25 mg DNA/ml) and then finally filtered with a 1.0 μm PES syringe filter. The filtered test article was pooled and diluted with D5W to the final injection dose (200μl per animal).
The compound or control was Intravenously administered on day 7 post-inoculation when measurable tumor growth was detected with an average tumor size of 150 mm 3 . Study mice were randomly assigned to each study group with an equal distribution of tumor size per group. Each group comprised 13 animals. 10 of the animals were enrolled in tumor growth inhibition study. Measurements of tumor volume (mm 3 ) were performed by digital calipers every 4 days for 40 days post tumor inoculation. Animal body weight (g) was measured in subcutaneous PANC1 xenografts on days 7, 14, 21, 28, 35, 39 after tumor inoculation (Day 0). Remaining 3 animals in each group were sacrificed at day 27 (two days post last infusion) from which tumors were harvested for molecular analysis.

Use of a dual luciferase reporter system to optimize KRAS mut mutant specific knockdown constructs
The psiCHECK2 reporter vector is a mammalian expression vector that expresses dual luciferase reporters on a single vector thereby allowing testing of two expressed sequences in the same environment under the same conditions. We inserted nucleotide sequences encoding the first 17 amino acids of KRAS into the regions encoding the amino terminal of the psi-CHECK2 vector luciferase reporter genes; i.e., the KRAS wt sequence was inserted into the renilla (RL) luciferase gene and a KRAS mut sequence was inserted into the firefly (FF) gene (see Fig 2A; for specific sequence insertion see S2A Fig). A total of five psiCHECK2-based test vectors were constructed: G12D, G12C, G12V, G12R, and one for the wild-type sequence only (S2A Fig). The test reporter constructs express a RL/FF ratio similar to the parent psiCHECK2 ( Fig 2B). A single nucleotide G!A change leads to G12D mutation. Using tiling approach, we constructed a series of bi-shRNA knockdown vectors with the G12D mutation's single nucleotide change positioned at positions 2-11 of the guide strand ( Fig 2C, panel a). We then cotransfected the G12D/WT dual expression test vector with the G12D specific knockdown vector and showed that positioning of the mutant sequence at different positions of the guide strand resulted in different FF/RL ratios ( Fig 2C, panel b). Slight variations in the test vector to knockdown vector ratio showed similar reproducible results (S2B Fig). All subsequent studies were done with the test vector to knockdown vector at 1 to 1 ratio. An advantageous knocked down mutant:wild-type ratio was obtained with complement to mutated nucleotide in positions 2, 3, 4, 5, 9, 10 and 11 of the guide strand. Complement to mutated nucleotide at positions 2, 3 or 4 of the guide strand were the most effective vis-a-vis selective mutant sequence knockdown not having a significant effect on wild-type transcripts, whereas substitutions at positions 7, 8, 9 and 11 reduced wild-type expression as well (S2C Fig). Position 3 and 4 substitutions for G12V, G12R and G12C were constructed and similarly tested; the comparative results are shown in Fig 2D. The results of the screening effort are summarized in S1 Table.

Triplex bi-shRNA-KRAS mut constructs can effectively and specifically knockdown KRAS mut expression without affecting KRAS wt expression in cultured cells
Given that the majority of oncogenic KRAS mutations are at codons 12 and 13, we designed a single transcription unit capable of a broadened range of KRAS mutant knockdown. Two sets of triplex knockdown vectors were constructed; one for G12D, G12V and G12R (51%, 30% and 12% of PDAC KRAS mutations, respectively) and another one for G12C, G12D and G12V (prevalent in colorectal and lung adenocarcinoma [17]). The most effective and discriminating of the G12D, G12V, G12C and G12R knockdown bi-shRNA cassettes were included in the triplex constructs; the guide strand location of each mutated nucleotide at positions 3, 4, 3 and 4, respectively. We also evaluated the polycistronic miR-17-92 cluster backbone [designated constructs 131 (bi-shRNA DVR ) and 132 (bi-shRNA CDV )] as an alternative to the miR-30a backbone [constructs 129 (bi-shRNA DVR ) and 130 (bi-shRNA CDV )] (schematically shown in Fig 3A; the sequences in S3A and S3B Fig). Using the dual reporter system, we demonstrated that all triplex constructs produced selective G12D, G12V, G12C and G12R knockdown albeit with varied efficiency (Fig 3B). For G12D and G12R, all four constructs were effective, although the latter was less so than the G12R specific bi-shRNA construct. Construct 131 was Coding sequences for the first 17 amino acids of KRAS wild-type (wt) and mutant (mu) were inserted into the amino terminus of the psiCHECK2's hRluc (renilla) and hluc (firefly) coding sequence, respectively. Knockdown of wt vs. mu sequence is compared by renilla to firefly intensity ratio. A. Schematic of the sequence insertion into psiCHECK2 for reporter constructs. B. Bar graph show comparison of reporter constructs relative light unit (RLU) intensity ratio of renilla (RL) to firefly (FF). Y-axis is RL/FF RLU ratio. X-axis is reporter test vectors and parent reporter vector. Standard deviation bar represents measurement from quadruplet samples of independently transfected cells in 96-well format and assayed simultaneously post transfection. C. Positional effect of G12D knockdown constructs; panel a: Table illustrate each constructs guide strand sequence in relation to G12D mutation site. 1 st column indicates position of G12D mutation in guide strand of each construct. 2 nd column is the code for each construct. The guide strand sequence is shown as the complement of target sequence at 3' to 5' orientation; panel b: Bar graph show comparative plot of FF/RL RLU ratio (mu/wt) for each knockdown construct. Sample C is the control without knockdown vector. The red bar represents average control sample value for visual enhancement. Standard deviation bar represents measurement from quadruplet samples of independently transfected cells in 96-well format and assayed simultaneously post transfection. Two-tailed student T-test indicates ρvalue 0.05 between control and samples 86, 87, 88, 75, 76 and 77. D. G12D, G12V, G12R and G12C knockdown constructs (with mutation nucleotide at position 3 or 4 of the guide strand) were tested against test reporter vectors of all four mutations. Bar graph shows the summary of relative average FF/RL RLU ratio (mu/wt). Ã indicate the most effective constructs for each mutation. X-axis is the knockdown constructs for G12D, G12V, G12R and G12C. P3 indicate knockdown construct with mutated nucleotide at position 3 of the guide strand. P4 indicate knockdown construct with mutated nucleotide at position 4 of the guide strand. Y-axis is the FF/RL RLU ratio. KRAS G12D heterozygous PANC1 cells were co-transfected with both knockdown and neomycin resistance expression vectors and then selected for G418-resistant stably transformed cells for Restriction Fragment Length Polymorphism (RFLP) assay to discriminate KRAS mut from KRAS wt (see S3C Fig). Non-transformed cells, empty vector cells and non-specific G12V knockdown vector transformed cells all showed mutant transcript comprising 80-84% of total KRAS transcripts ( Fig 3C, lanes 1, 2 and 7). There was proportionally less mutant transcript (63-70%) with constructs 129 and 130 (Fig 3C, lanes 3 and 4), whereas constructs 131 and 132 reduced the mutant transcript proportion to 9-12% of the total or 10.7-14.3% of that seen with the empty vector mutant (Fig 3C, lanes 5 and 6). Interestingly, the total amount of KRAS transcript (mutant + wild-type) was the same in control and knockdown cells.

Position specific bi-shRNA-KRAS mut constructs reduce PANC1 cell growth in vitro
Pancreas ductal adenocarcinoma cell PANC1 with KRAS G12D/wt , human embryonic kidney cell HEK293 with KRAS wt/wt and colorectal cancer cell HT29 with KRAS wt/wt were tested for growth inhibition in vitro. In so far as high doses non-discriminatively inhibited cell growth presumably due to non-specific transfection effect, we determined 10  Triplex bi-shRNA-KRAS mut constructs effectively reduce tumor xenograft growth in vivo 1x10 6 PANC1 cells were subcutaneously implanted in female athymic Nu/Nu mice and treatment started when tumor volume reached 150 mm 3 . Based on in vitro activity (Fig 3C), 5 μg or 25 μg per infusion of the bi-shRNA KRAS fusogenic lipoplex constructs 131 or 132 were administered via slow tail vein injection twice weekly for four weeks. The lipoplex formulation was freeze-dried and stored at 4˚C, then reconstituted and filtered prior to each application. Tumor growth was inhibited by both constructs in a dose-dependent manner, with 132 being the most effective (Fig 4A). Treatments were well tolerated and weight loss was not observed. Tumor sampling confirmed intratumoral plasmid delivery and copy number correlation with administered dose (Fig 4B). We postulate that the discrepancy in delivery and consequent decreased growth inhibition of construct 131 resulted from the re-constitution and filtration process of the freeze-dried formulation (Fig 4A, groups 3 and 4).

Treated tumor samples show KRAS mutant specific knockdown and activation of EGFR signaling in vivo
Tumors were sampled from each treatment group at two days after the sixth treatment. Tumors were preserved with Qiagen AllProtect after harvesting and subsequently stored at -20˚C before processing for molecular analysis. One half of each preserved tumor was analyzed by RFLP to determine the wild-type to mutant KRAS mRNA transcript ratio. Empty liposome treated group 2 and construct 132 lipoplex (S3B Fig) treated groups 5 and 6, were examined and compared ( Fig  5A). The WT/mutant transcript ratio of the empty liposome control treated group was approximately 0.25 (20%/80%); very much the same as for the PANC1 cells in vitro (Fig 3C). The ratios in treatment group 5 (construct 132, 5 μg) ranged from 0.57 (36.14%/63.86%) to 1.9 (65.68%/ 34.32%); in the higher dose group (group 6; construct 132, 25 μg) the ratios were 3.6 (78.37%/ 21.63%), 5.8 (85.23%/14.77%) and no detectable mutant transcripts in sample 6A.
KRAS mut basal signaling through the RAF/MEK/ERK pathway differentially regulates EGFR tyrosine and threonine autophosphorylation resulting in negative feedback control as demonstrated in a variety of KRAS mutated tumors [18][19][20][21][22]. Therefore, we examined the in vivo PANC1 mouse xenograft tumors (used for RFLP) to document the pattern of EGFR autophosphorylation (Western immunoblot). The total EGFR expression level in treated tumors averaged less than a 2-fold difference compared to untreated tumors (Fig 5B, panel c). On the other hand, in the construct 132 treated group there was a significant increase in the activating Y1068 EGFR phosphorylation site (Fig 5B, panel b) as well as in EGFR tyrosine phosphorylation sites Y1045, Y1068 and Y1125 (Fig 5C and S5A Fig). We also examined MEK, ERK and AKT expression and phosphorylation (S5B Fig). pMEK and pERK were somewhat higher in higher dose treated tumors (S5C Fig, panel a, treatment groups 4 and 6) but without change in pAKT at S473 and only slightly lowered at site T308 (S5D Fig). Protein levels for ERK and AKT were slightly lower (S5D Fig). Differing from most reported in vitro knockdown studies [7,[23][24][25], total RAS protein expression was about the same for treated versus untreated group (Fig 5B, panel a). The absence of non-target KRAS wt knockdown minimizes the risk of toxicity.

Discussion
The overall mutational frequency of KRAS in cancer is 22% but with non-uniform distribution amongst different cancer types. Greater than 90% of PDAC carry KRAS mutations (with a relatively high mutant allele specific imbalance) and the mutation frequency in lung and colorectal cancer are approximately 30-50% and 40-50%, respectively (COSMIC database). KRAS isoform specific mutation frequency likewise varies with cancer type, as do downstream signaling processes. This report demonstrates a novel purposefully designed KRAS multi-mutation genotype specific knockdown moiety with the potential for a systemically delivered therapeutic approach in a majority of patients with pancreas cancer. Previous studies with PC-7 (KRAS G12V/G12V ) and PANC1 (KRAS G12D/wt ) xenografts have shown that siRNA vectors targeting KRAS codon-12 mutations can be effective in reducing tumor growth when injected intratumorally [26]. Others have extended this approach to humans by combining an intratumoral siRNA vector with systemic chemotherapy to treat locally advanced, unresectable pancreatic cancers [27]. This was shown to be safe, well tolerated and with preliminary demonstration of clinical benefit; i.e. prolonged tumor control, shrinkage and biomarker reduction. However, given the high rate of early metastagenicity of pancreatic cancer and the advanced stage at diagnosis, local intratumoral injection of siRNA vectors is not a viable option for the majority of PDAC patients. AZD4785, an antisense oligonucleotide that indiscriminately targets KRAS mut and KRAS wt achieves effective mutant and wild-type knockdown both in vitro and in vivo by systemic delivery without demonstrable adverse effects [28]. Although the authors suggest that NRAS and/or HRAS compensate for KRAS wt knockdown, it remains unclear whether or not the compartmentalization and functional specificity of the RAS isoforms will allow effective and safe clinical translation of this approach [29][30][31].
Both siRNA-and shRNA-mediated target gene expression knockdown have been shown to distinguish single nucleotide differences between alleles [32][33][34]. The position of the single nucleotide difference on the guide strand and the type of nucleotide matches are important factors determining efficacy. For siRNA, Schwarz et al. have shown that mismatches at guide strand positions 5, 9, 10, 12, 13 and 16 were most effective for differential expression knockdown, and that purine to purine mismatches are more discriminating than other mismatch types [32]. Specificity differences due to nucleotide positioning on the guide strand are possibly ascribable to target sequence composition, use of siRNA vs. shRNA, and/or different assay methods [32][33][34][35]. Rather than the seed region (proposed sequence recognition region), the central region, which contains the cleavage site for cleavage-dependent RNAi, was the most consistent site for sequence selectivity. Almost all KRAS mutant specific knockdown publications place the mutant sequence at the central region [23-25, 27, 36]. In addition to the central region, positions 4 and 16 of the guide strand were also found to be useful in sequence distinction [34,35,37]. Insofar as bi-shRNA-KRAS mut utilizes both cleavage-dependent and cleavageindependent mechanisms, we decided to use the systematic tiling strategy to interrogate the positional effect that would result in the most effective, discriminatory, mutant-specific KRAS knockdown. Unlike most assay systems used by others that either attach the target sequence at the 3' end of a reporter gene or attach the reporter sequence at the carboxyl terminus of a target gene or in polycistronic fashion, we inserted the KRAS coding sequence of the first 17 amino acids at the amino terminus of reporter genes to mimic the natural target gene sequence location with respect to both transcription and translation. Additionally, we placed both wild-type and mutant sequences on one dual luciferase reporter expression vector in order to compare WT and mutant knockdown in the same cell and environment. The insertion of the target sequence at the amino terminus did not affect the reporter gene expression. Positions 3 and 4 of the guide strand in the seed region rather than in the central region were the most discriminating. Notably, our in vitro growth inhibition study found placing the mutated nucleotide at the central region not only affected KRAS G12D mutation cells (PANC1), but also KRAS wt cells (HEK293). That there was no growth effect for KRAS wt HT29 cells may due to inefficient in vitro transfection. The results from reporter assays were successfully translated to both in vitro and in vivo studies that showed effective knockdown of the mutant transcript without affecting the wild-type transcript in a native environment.
Onco-relevant RAS downstream signaling is complex and appears to be primarily mediated via three effector pathways: 1) Raf-MEK-ERK, 2) PI3k-AKT-mTOR, and 3) RalGEF-Ral with extensive pathway cross talk and regulatory feedback pathways [18][19][20][21][22]. Pathway utilization patterns have been shown to be mutation specific. For example, NSCLC cell lines with KRAS G12D show activated PI3k and MEK whereas those with KRAS G12C and KRAS G12V show activated Ral and decreased AKT [38]. In addition, pathway utilization patterns are also tumor type context specific. MEK/ERK inhibition in lung adenocarcinoma lines with KRAS G12C decreases EZH2 expression but is without affect in cell lines with KRAS G12V . On the other hand, MEK/ERK inhibition decreases EZH2 expression in colon and pancreatic cancer lines with both KRAS G12C and KRAS G12V [39]. There are also data indicating the potential therapeutic relevance of regulatory feedback systems within these pathways. As an example, constitutively active KRAS mut regulates basal MEK signaling [7,40], which in turn has a negative feedback effect on EGFR activity by enhancing inhibitory phosphorylation (e.g., T669) and relieving activating phosphorylation (e.g., Y1068, Y1069, and Y1125) at functionally specific binding sites [14,24,40]. Consequently, KRAS mut downregulation facilitates activated EGFRmediated RAS wt -GDP!RAS wt -GTP configuration as seen in mut/WT cell lines and increases NRAS-GTP levels in mut/WT and mut/-cell lines [14,40]. In addition to being heteroallelic (KRAS G12D/wt ) PANC1 cells exhibit higher EGFR copy number than other pancreatic cancer cell lines (PANC1 > MIA PaCa-2 > Capan-2). Data from our in vivo mutant-specific bi-shRNA treated PANC1 tumors show significantly increased p-EGFR with phosphorylation at Y1068 (Y1069), Y1045 and Y1125 sites, compared to vehicle treated or untreated tumors, without significant changes in total EGFR protein (clearly demonstrating enhanced tyrosine kinase activity due to post-translational modification induced activation rather than to increased protein expression). These data, as well as significant differential gene expression patterns (which will be presented in a separate paper) confirm that expression of the mutant KRAS allele was effectively and specifically suppressed. The consequent reactivation of EGFR signaling suggests a potential therapeutic benefit from combinatorial bi-shRNA KRAS mut and EGFR inhibition. The RFLP data reveal that whereas the stable KRAS mut mRNA was significantly repressed in treated tumor and PANC1 cells in vitro the KRAS wt mRNA population is proportionally increased. These RFLP mRNA data are consistent with the lack of effect of our knockdown constructs on total KRAS protein, e.g., group 6 in Fig 5B, panel a, despite > 80% suppression of the mutant KRAS allele (Fig 5A). These findings differ from other KRAS mutant specific knockdown studies that show reduction of total KRAS protein [23][24][25] as well as reduction of both stable KRAS wt and KRAS mut mRNA [24] and, therefore, require further investigation. Insofar as the commonly used anti-RAS antibodies do not differentiate between RAS protein isoforms it may be that suppression of KRAS mut expression relieves the negative feedback KRAS mut !NRAS (and, possibly, both HRAS and NRAS) pathway[s] [40] thereby stabilizing total RAS expression. Other potentially contributory mechanisms to consider are 1) differences between studies in procedures and xenograft genotypes/phenotypes and their derivative signaling pathways [41], 2) the presence of extensive intratumoral heterogeneity as seen in recent CTC single cell expression data [42], 3) a lower KRAS mut ratio [43], 4) a KRAS mut effect on stem cell distribution (which would also account for epithelial-mesenchymal (EMT) shift) [44,45], and 5) the stochastic, non-determinant loss of KRAS mut stem cells [44].
A major obstacle thus far preventing translation of RNAi technology to the clinic has been lack of effective systemic delivery comprising distribution, metabolism/elimination and tissue/ intracellular entry. Using a non-targeted, fusogenic lipoplex [46,47], we show that payload plasmid DNA is effectively delivered to tumors in vivo in functionally adequate concentrations. Although a freeze-dried formulation has the advantage of long-term stability allowing for storage and transport, heterogeneity and inconsistency in reconstituted material is problematic. In our experience the physical properties of the reconstituted material vary from batch to batch, often resulting in larger aggregates of test agents which require an additional filtration process to eliminate large particles (1, panel A). This makes it difficult to determine the concentration of the actual delivered final product. Additionally, batch-to-batch variation is difficult to control. In our initial tests of constructs 131 and 132, in vitro (PANC-1) studies showed both constructs capable of selective knockdown of the KRAS mutant allele expression without affecting wild-type allele whereas the in vivo study showed that construct 131 was not as effective as 132. Subsequent analysis of intratumoral DNA revealed that 131 was not as efficiently delivered as 132, supporting the inconsistency of the freeze-dried formulation. We have since developed a new lipoplex formulation process using the ethanol injection method and are in the process of completing optimization studies prior to GMP product manufacturing and large animal toxicology studies. The resulting product has a narrower volumetric range with a smaller average diameter, homogenous physical properties (Table 1, panel B), an efficient delivery and a process that is scalable. The modular, multi mutant-specific bi-shRNA KRAS herein described represents a unique therapeutic approach to cancer, including those with multiple mutant heteroalleles and/or those with two or more synthetic lethals. The safety and biodistributiion of the systemically delivered fusogenic lipoplex is currently being evaluated in a phase I clinical trial of bi-shRNA EWS/FLI1 in patients with Ewing's sarcoma (BB-IND 16939). Insofar as the lack of KRAS mut druggable sites, the multifarity of downstream signaling pathways and the lack of a safe, efficient, and systemic tumor selective delivery vehicle have stymied the development of a translatable targeted treatment for KRAS mutated cancers, the bi-shRNA KRAS lipoplex, by addressing these obstacles, is primed for clinical implementation.