The clinical development of tyrosine kinase inhibitors (TKI) has led to great strides in improving the survival of chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) patients. But even the new generation TKIs are rendered futile in the face of evolving landscape of acquired mutations leading to drug resistance, necessitating the pursuit of alternative therapeutic approaches. In contrast to exploiting proteins as targets like most conventional drugs and TKIs, RNA Interference (RNAi) exerts its therapeutic action towards disease-driving aberrant genes. To realize the potential of RNAi, the major challenge is to efficiently deliver the therapeutic mediator of RNAi, small interfering RNA (siRNA) molecules. In this study, we explored the feasibility of using aliphatic lipid (linoleic acid and lauric acid)-grafted polymers (lipopolymers) for the delivery of siRNAs against the FLT3 oncogene in AML and BCR-ABL oncogene in CML. The lipopolymer delivered siRNA potently suppressed the proliferation AML and CML cells via silencing of the targeted oncogenes. In both AML and CML subcutaneous xenografts generated in NCG mice, intravenously administered lipopolymer/siRNA complexes displayed significant inhibitory effect on tumor growth. Combining siFLT3 complexes with gilteritinib allowed for reduction of effective drug dosage, longer duration of remission, and enhanced survival after relapse, compared to gilteritinib monotherapy. Anti-leukemic activity of siBCR-ABL complexes was similar in wild-type and TKI-resistant cells, and therapeutic efficacy was confirmed in vivo through prolonged survival of the NCG hosts systemically implanted with TKI-resistant cells. These results demonstrate the preclinical efficacy of lipopolymer facilitated siRNA delivery, providing a novel therapeutic platform for myeloid leukemias.
Aniline Compounds , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Leukemia, Myeloid, Acute , Pyrazines , Humans , Animals , Mice , RNA, Small Interfering , Fusion Proteins, bcr-abl/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Oncogenes , Models, Animal , Protein Kinase Inhibitors/therapeutic use , Protein Kinase Inhibitors/pharmacology , Drug Resistance, Neoplasm
The past two decades have witnessed significant strides in leukemia therapies through approval of therapeutic inhibitors targeting oncogene-driving dysregulated tyrosine kinase activities and key epigenetic and apoptosis regulators. Although these drugs have brought about complete remission in the majority of patients, many patients face relapse or have refractory disease. The main factor contributing to relapse is the presence of a small subpopulation of dormant drug-resistant leukemia cells that possess stem cell features (termed as leukemia stem cells or LSCs). Thus, overcoming drug resistance and targeting LSCs remain major challenges for curative treatment of human leukemia. Chronic myeloid leukemia (CML) is a good example, with rare, propagating LSCs and drug-resistant cells that cannot be eradicated by BCR-ABL-directed tyrosine kinase inhibitor (TKI) monotherapy and that are responsible for disease relapse/progression. Therefore, it is imperative to identify key players in regulating BCR-ABL1-dependent and independent drug-resistance mechanisms, and their key pathways, so that CML LSCs can be selectively targeted or sensitized to TKIs. Here, we describe several easily adaptable gene knockdown approaches in CD34+ CML stem/progenitor cells that can be used to investigate the biological properties of LSCs and molecular effects of genes of interest (GOI), which can be further explored as therapeutic modalities against LSCs in the context of human leukemia.
Fusion Proteins, bcr-abl , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Drug Resistance, Neoplasm/genetics , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Gene Knockdown Techniques , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Neoplastic Stem Cells , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Recurrence
The discovery of RNA interference (RNAi) more than two decades ago opened avenues for avant-garde cancer treatments that possess the ability to evade issues hampering current chemotherapeutic strategies, owing to its specific gene sequence-driven mechanism of action. A potent short interfering RNA (siRNA) delivery vehicle designed to overcome physiological barriers is imperative for successful RNAi therapy. For this purpose, this study explored the characteristics and therapeutic efficacy of low-molecular weight (MW) polyethylenimine (PEI) with high cholesterol substitution, yielding water-insoluble polymers, in chronic myeloid leukemia (CML) K562 cells. A strong impact of cholesterol grafting on the physicochemical attributes of the resultant polymers and their corresponding complexes with siRNA was observed, with the siRNA binding capacity of polymers increasing and complex dissociation sensitivity decreasing with increase in cholesterol content of the polymers. The modified polymer complexes were significantly smaller in size and possessed higher cationic charge compared to the parent polymer. The interaction with anionic heparan sulfate preoteoglycans present on the cell surface was significant in cellular uptake of the complexes. The therapeutic efficacy of siRNA/polymer complexes was reflected in their ability to effectively silence the reporter green fluorescent protein gene and endogenous CML oncogene BCR-ABL as well as significantly inhibit colony formation by K562 cells post BCR-ABL silencing. The results of this study demonstrated beneficial effects of high levels of hydrophobic substitution on low MW PEI on their functional performance bestowing them the potential to be potent RNAi agents for CML therapy.
Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Polyethyleneimine , RNA Interference , RNA, Small Interfering , Humans , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Polyethyleneimine/chemistry , Polyethyleneimine/pharmacology , RNA, Small Interfering/chemistry , RNA, Small Interfering/pharmacology
Anchorage-independent cells possess morphological features and cell membrane compositions that are distinct from adherent cells. They display minimal surface area, have a low rate of endocytosis and generally possess few proteoglycans which make it a challenge to deliver nucleic acids into them. Wide ranges of methods and materials have been developed to tackle the delivery obstacles for the polynucleotide-based therapeutics in modifying non-adherent cells. This article summarizes the techniques and biomaterials that have been utilized for transfection of anchorage-independent cells. First, physical techniques are briefly described along with particular applications for which they are well-suited. The structure-activity relationship of various biomaterial carriers of polynucleotides are then discussed with strategies employed to enhance their capability to transfect anchorage-independent cells. In conclusion, the authors' perspectives on different methods for polynucleotide delivery to primary human cells are compared, along with a discussion of their progression towards clinical trials.
