A major challenge with the use of siRNAs in mammals is their intracellular delivery to specific tissues and organs that express the target gene. The first demonstrations of siRNA-mediated gene silencing in mammals through systemic administration were accomplished using naked siRNA and methods of administration not compatible with clinical application.5–7
Since then, several delivery vehicles have been combined with siRNAs to improve their delivery in animal models.1,2
Soutschek et al.
were the first to provide direct evidence for the siRNA mechanism of action by using a modified 5’-RACE (rapid amplification of cDNA ends) PCR technique providing positive identification of the specific mRNA cleavage product.8
Human clinical trials with synthetic siRNAs began in 2004, utilizing direct intraocular siRNA injections for patients with blinding choroidal neovascularization (CNV). Subsequently, other clinical trials have initiated2
and early clinical data are beginning to appear.9,10
While there are animal studies that do support the possibility of an RNAi mechanism of action from administered siRNA,11
other animal data from siRNAs injected into the eyes of mice for the treatment of CNV suggest non-RNAi mechanisms of action for CNV suppression.12
At this time, no direct evidence for an RNAi mechanism of action in humans from siRNA administered either locally or systemically has been reported.
We are currently conducting the first siRNA clinical trial that utilizes a targeted nanoparticle delivery system (clinical trial registration number NCT00689065).13
Patients with solid cancers refractory to standard-of-care therapies are administered doses of targeted, nanoparticles on days 1, 3, 8 and 10 of a 21-day cycle via a 30-minute i.v. infusion. The nanoparticles consist of a synthetic delivery system containing (): (i) a linear, cyclodextrin-based polymer (CDP), (ii) a human transferrin protein (hTf) targeting ligand displayed on the exterior of the nanoparticle to engage Tf receptors (hTfR) on the surface of the cancer cells, (iii) a hydrophilic polymer (polyethylene glycol (PEG) used to promote nanoparticle stability in biological fluids), and (iv) siRNA designed to reduce the expression of the M2 subunit of ribonucleotide reductase (RRM2: sequence used in the clinic was previously denoted siR2B+5).14
The TfR has long been known to be up-regulated in malignant cells,15
and RRM2 is an established anti-cancer target.16
These nanoparticles (clinical version denoted as CALAA-01) have been shown to be well tolerated in multi-dosing studies in non-human primates.17
While a single patient with chronic myeloid leukemia has been administered siRNA via liposomal delivery,18
our clinical trial is the initial human trial to systemically deliver siRNA with a targeted delivery system and to treat patients with solid cancer.13
Figure 1 Detection of targeted nanoparticles in human tumors. (a). Schematic representation of the targeted nanoparticles. The polyethyleneglycol (PEG) molecules are terminated with adamantane (AD) that form inclusion complexes with surface cyclodextrins in order (more ...)
In order to ascertain whether the targeted delivery system can provide effective delivery of functional siRNA to human tumors, we investigated biopsies from three patients from three different dosing cohorts; patients A, B and C, all of whom had metastatic melanoma and received doses of CALAA-01 of 18, 24 and 30 mg-siRNA/m2, respectively. Given the highly experimental nature of this protocol, the regulatory process at both the local and federal levels explicitly precluded a provision for mandatory biopsies in all patients. Therefore, biopsies were obtained on a voluntary basis. Biopsies in these three patients were collected after the final dose of cycle 1 (denoted Apost, Bpost and C1post) and compared to archived tissue (denoted Apre, Bpre and C1pre). Patient C continued on therapy beyond one cycle and provided another set of biopsy materials (C2pre that was obtained approximately one month after the final dose of cycle 1 and C2post that was collected on the day of the final dose of cycle 2). Because of limited sample amount, only immunohistochemistry (IHC) and staining for the nanoparticles could be performed on the C1pre and C1post samples, and protein (for Western blot analyses) was only available from the C2pre and C2post samples. Details of this clinical trial will be reported elsewhere when completed.
The targeted nanoparticles (ca. 70 nm diameter) were administered i.v., as they are designed to circulate and then to accumulate and permeate in solid tumors.13
Within the tumor, the hTf targeting ligand assists in directing the nanoparticles into tumor cells overexpressing hTfR.19
To detect the nanoparticles in tumor cells, sections of the tumor tissue were stained for the presence of the nanoparticles using a 5 nm gold particle that is capped with thiolated PEG containing adamantane (AD) at the end distal to the thiol (AD-PEG-Au) to allow for multivalent binding to the cyclodextrins (Supplementary Scheme SI 2
). The function of the stain has been previously confirmed using other cyclodextrin-containing particles,20
and is demonstrated here for the targeted nanoparticles carrying siRNA in vitro
(Supplementary Fig. 1
) and in vivo
(Supplementary Figs. 2 and 3
). Transmission electron microscopy (TEM) images of the nanoparticles confirm that in mice, the nanoparticles are intracellular (Supplementary Fig. 2
). Samples A, B and C1, analyzed in a blinded fashion, demonstrated a heterogeneous distribution of nanoparticles only in post-dosing tumor tissue ( for post-dosing and Supplementary Fig. 4
for pre-dosing). The nanoparticles can localize intracellularly in tumor tissue and are not found in the adjacent epidermis (). In these biopsies TEM images were dominated by melanosomes21
inhibiting the identification of the nanoparticles (data not shown). Samples C1post
reveal the highest number and intensity of stained regions, Bpost
exhibits a decreased amount of staining relative to samples C1post
does not reveal the presence of the stain (), and all the pre-dosing samples are completely negative for the stain (Supplementary Fig. 4
). This is the first example of a dose-dependent accumulation of targeted nanoparticles in tumors of humans from systemic injections for nanoparticles of any type.
Tumor RRM2 mRNA levels were measured by quantitative real time polymerase chain reaction (qRT-PCR) and were performed in a blinded fashion.22
Reduction in RRM2 mRNA is observed in the post-treatment samples (). Since samples Apre
are from tissues collected many months before the initiation of siRNA treatment, the fraction of the overall reduction in mRNA observed in Apost
attributable to the nanoparticle treatment cannot be directly ascertained. Unfortunately, we were not able to perform PCR on the C1 samples. However, the PCR data from the C2pre
samples (collected 10 days apart) provide direct evidence for RRM2 mRNA reduction via the treatment of the patient with the nanoparticles.
Figure 2 RRM2 mRNA and protein expression in tumor tissue. (a). qRT-PCR analysis of RRM2 mRNA levels in samples from patients A and B before and after dosing. RRM2 mRNA levels are normalized to TBP mRNA levels. Results are presented as percentage of the pre-dosing (more ...)
To ascertain whether the RRM2 protein level is reduced in the tumor because of the siRNA treatment, IHC and Western blotting were employed as previously described in mice.23
Since RRM2 protein expression is largely restricted to the late G1/early S phase of cell cycle, not all of the tumor cells will be expressing RRM2. shows IHC data for RRM2 and TfR proteins in C1pre
samples (IHC analyses were performed in a blinded fashion and 10 random regions of each sample were analyzed). Significant reduction in RRM2 is observed (mean scoring of RRM2 from the 10 sections was reduced 5-fold) after treatment while TfR levels are somewhat elevated (mean scoring of TfR from the 10 sections was increased 1.2-fold) in the C1pre
samples. The low level of RRM2 that is observed by IHC in the C1post
sample is maintained in the C2pre
samples (by IHC). Western blot analyses of the C2pre
samples reveal a reduction in the level of the RRM2 protein that is due to the siRNA treatment (RRM2 mRNA reductions exceeded the reduction levels obtained from protein but this could be due to post-transcriptional mechanisms that have been observed previously24
). The decreases in the RRM2 mRNA and protein observed after treatment () suggest the siRNA treatment remains effective after several cycles of dosing. The IHC data from patient A do not reveal changes in RRM2 expression after dosing, while results from patient B are indicative of reductions in maximal RRM2 expression (IHC scoring of the regions of maximal expression showed a 1.5-fold decrease) but the overall mean expression levels remained relatively constant (IHC scoring of the 10 sections).
Figure 3 Ribonucleotide reductase (RRM2) and tranferrin receptor (TfR) protein expression in C1pre and C1post samples. Photomicrographs of malignant melanoma belonging to a, b, c, pre-treatment and d, e, f post-treatment samples. Protein expression is represented (more ...)
To demonstrate that the siRNA delivered via the targeted nanoparticles can engage the RNAi machinery, the mRNA cleavage products were characterized using a modified 5’-RNA ligand-mediated rapid amplification of cDNA ends (5’-RLM-RACE) PCR technique (). A RRM2 mRNA fragment, whose 5’ end matches the predicted cleavage site (10 base pairs from the 5’ end of the antisense strand), was detected in the C2pre
samples, but not from Bpost
, and Apost
or their corresponding pre-treatment samples. RACE does not provide a quantitative measure of the amount of the fragments so the intensities of the bands cannot be correlated with amounts in the tissue samples. The presence of this RRM2 mRNA fragment from patient C indicates siRNA delivered via targeted nanoparticles can engage the RNAi machinery in a solid tumor of a human and induce the desired mRNA cleavage. Furthermore, this result suggests that at least a portion of the RRM2 mRNA and protein reductions observed from the C2 samples are due to a bona fide RNAi mechanism. The presence of the RRM2 mRNA fragment in the C2pre
sample suggests that siRNA can provide an RNAi mechanism for several weeks (mRNA cleavage in the C2pre
sample must originate from cycle one dosing) as the RRM2 protein levels remained relatively constant when compared to the C1post
sample (IHC). We have shown that the length of the RNAi effects of delivered siRNA in both cells and animals (mice) is dependent on the doubling time of the cells being analyzed (longer inhibition times with longer cell doubling times).25
Gene silencing by siRNA can occur on the timescale observed here, ca. one month, provided the cell doubling times are long.25
Patient C had stable disease between these biopsies, and these mostly quiescent tumors have very slow growth kinetics that would be suitable to experience lengthy RNAi effects.25
Additionally, we do not know how long the nanoparticles reside within the cells and release siRNA. Since the nanoparticles are observed in the sample C1post
and not the sample C2pre
, they must disassemble within one month. Thus, the pharmacodynamics of the RNAi effects could be due to the combination of the nanoparticle disassembly time and the time that the siRNA resides within the RNAi machinery.
Figure 4 5’-RLM-RACE detection of siRNA induced mRNA cleavage fragment. (a) Schematic depicting the location of the predicted anti-RRM2 siRNA cleavage site and the primers used for PCR amplification of the cleavage fragment. (b) Agarose gel of 5’-RLM-RACE (more ...)
The data presented here when taken together provide the first mechanistic evidence of RNAi in a human from an administered siRNA. Moreover, these data demonstrate the first example of dose dependent accumulation of targeted nanoparticles in human tumors. The reduction of the RRM2 mRNA and protein by the RRM2-specific siRNA is observed, and the results from 5’-RLM-RACE analyses reveal that the delivered siRNA engages the RNAi machinery. These data demonstrate that RNAi can occur in a human from a systemically delivered siRNA, and that siRNA can be used as a gene-specific therapeutic.