Trimeric Small Interfering RNAs and Their Cholesterol-Containing Conjugates Exhibit Improved Accumulation in Tumors,
Ivan V Chernikov1, Daniil V Gladkikh1, Ulyana A Karelina1
1Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk 630090, Russia.
Abstract:
Cholesterol derivatives of nuclease-resistant, anti-MDR1 small-interfering RNAs were designed to contain a 2'-OMe-modified 21-bp siRNA and a 63-bp TsiRNA in order to investigate their accumulation and silencing activity in vitro and in vivo. The results showed that increasing the length of the RNA duplex in such a conjugate increases its biological activity when delivered using a transfection agent. However, the efficiency of accumulation in human drug-resistant KB-8-5 cells during delivery in vitro in a carrier-free mode was reduced as well as efficiency of target gene silencing. TsiRNAs demonstrated a similar biodistribution in KB-8-5 xenograft tumor-bearing SCID mice with more efficient accumulation in organs and tumors than cholesterol-conjugated canonical siRNAs; however, this accumulation did not provide a silencing effect. The lack of correlation between the accumulation in the organ and the silencing activity of cholesterol conjugates of siRNAs of different lengths can be attributed to the fact that trimeric Ch-TsiRNA lags mainly in the intercellular space and does not penetrate sufficiently into the cytoplasm of the cell. Increased accumulation in the organs and in the tumor, by itself, shows that using siRNA with increased molecular weight is an effective approach to control biodistribution and delivery to the target organ.
Insights
Cholesterol-conjugated small interfering RNAs (siRNAs) and longer trimeric siRNAs (TsiRNAs) were studied for drug resistance gene silencing. Longer RNA duplexes improved delivery with agents but reduced silencing without them, suggesting improved biodistribution doesn't guarantee cellular uptake.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Multidrug resistance (MDR) mediated by the MDR1 gene poses a significant challenge in cancer therapy.
- Small interfering RNAs (siRNAs) offer a promising strategy for gene silencing, but their delivery and efficacy require optimization.
- Cholesterol conjugation and modified RNA structures are explored to enhance siRNA stability, delivery, and therapeutic potential.
Purpose of the Study:
- To design and evaluate cholesterol derivatives of nuclease-resistant anti-MDR1 small interfering RNAs (siRNAs) and trimeric siRNAs (TsiRNAs).
- To investigate the in vitro and in vivo accumulation and gene silencing activity of these modified RNA constructs.
- To determine the correlation between RNA accumulation, biodistribution, and therapeutic efficacy in drug-resistant cancer models.
Main Methods:
- Synthesis of 2'-OMe-modified 21-bp siRNA and 63-bp TsiRNA cholesterol conjugates.
- In vitro evaluation of cellular accumulation and gene silencing in human drug-resistant KB-8-5 cells.
- In vivo biodistribution studies in SCID mice bearing KB-8-5 xenografts.
- Assessment of target gene (MDR1) silencing efficacy in vitro and in vivo.
Main Results:
- Increasing RNA duplex length in conjugates enhanced biological activity with transfection agents.
- Carrier-free delivery in vitro showed reduced accumulation and silencing efficiency for longer constructs.
- Cholesterol-conjugated TsiRNAs exhibited improved organ and tumor accumulation in vivo compared to canonical siRNAs.
- Despite enhanced accumulation, TsiRNAs did not yield significant MDR1 gene silencing, potentially due to insufficient cytoplasmic penetration.
Conclusions:
- Cholesterol conjugation and increased RNA length can effectively control biodistribution and enhance organ/tumor accumulation.
- Enhanced accumulation alone does not guarantee therapeutic efficacy; cellular uptake and cytoplasmic delivery are critical.
- The findings highlight the complex interplay between RNA structure, delivery method, and biological activity for siRNA-based therapeutics.
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