Progress in RNAi-mediated Molecular Therapy of Acute and Chronic Myeloid Leukemia
2015; Cell Press; Volume: 4; Linguagem: Inglês
10.1038/mtna.2015.13
ISSN2162-2531
AutoresBreanne Landry, Juliana Valencia‐Serna, Hilal Gül-Uludağ, Xiaoyan Jiang, Anna Janowska‐Wieczorek, Joseph Brandwein, Hasan Uludağ,
Tópico(s)Virus-based gene therapy research
ResumoLeukemias arise from genetic alterations in normal hematopoietic stem or progenitor cells, leading to impaired regulation of proliferation, differentiation, apoptosis, and survival of the transformed cells. With the advent of RNA interference (RNAi) and the short interfering RNA (siRNA) as its pharmacological mediator, it is becoming possible to modulate specific targets at will. This article summarizes current attempts to utilize RNAi reagents for therapy of leukemias, focusing on acute and chronic myeloid leukemia. We first present unique aspects of RNAi-mediated therapy, followed by a brief background on the delivery technology of RNAi reagents. The need for leukemia-specific delivery of siRNA is discussed by describing approaches that targeted agents to leukemic cells. Pharmacokinetics and biodistribution of RNAi agents are then presented, highlighting the critical issues pertinent to emerging siRNA therapy. Efforts to deliver specific RNAi therapies are then summarized in the context of expected clinical outcomes, focusing on limiting leukemic cell survival, sensitizing malignant cells to chemotherapy, mobilization of leukemic cells, and eradication of leukemic stem cells. We conclude with a perspective on the future of RNAi therapy, emphasizing the technological requirements and mechanistic challenges for clinical entry. Leukemias arise from genetic alterations in normal hematopoietic stem or progenitor cells, leading to impaired regulation of proliferation, differentiation, apoptosis, and survival of the transformed cells. With the advent of RNA interference (RNAi) and the short interfering RNA (siRNA) as its pharmacological mediator, it is becoming possible to modulate specific targets at will. This article summarizes current attempts to utilize RNAi reagents for therapy of leukemias, focusing on acute and chronic myeloid leukemia. We first present unique aspects of RNAi-mediated therapy, followed by a brief background on the delivery technology of RNAi reagents. The need for leukemia-specific delivery of siRNA is discussed by describing approaches that targeted agents to leukemic cells. Pharmacokinetics and biodistribution of RNAi agents are then presented, highlighting the critical issues pertinent to emerging siRNA therapy. Efforts to deliver specific RNAi therapies are then summarized in the context of expected clinical outcomes, focusing on limiting leukemic cell survival, sensitizing malignant cells to chemotherapy, mobilization of leukemic cells, and eradication of leukemic stem cells. We conclude with a perspective on the future of RNAi therapy, emphasizing the technological requirements and mechanistic challenges for clinical entry. Limitations of Current Leukemia Therapies and Promise of RNAiLeukemic cancers arise from genetic alterations in normal hematopoietic stem or progenitor cells, leading to impaired regulation of proliferation, differentiation, and apoptosis as well as survival of malignant cells. Approximately 350,000 people worldwide are diagnosed with leukemia annually, leading to ∼250,000 deaths each year. An overall 5-year relative survival rate of 56.0% (between 2003 and 2009) is estimated for various leukemias combined.1National Cancer Institute Surveillance Research Program Surveillence RP Fast Stats: An interactive tool for access to SEER cancer statistics. 2013Google Scholar The front-line therapy in leukemia is chemo (drug) therapy,2Estey EH Acute myeloid leukemia: 2013 update on risk-stratification and management.Am J Hematol. 2013; 88: 318-327Crossref PubMed Scopus (0) Google Scholar,3Stefanachi A Leonetti F Nicolotti O Catto M Pisani L Cellamare S et al.New strategies in the chemotherapy of leukemia: eradicating cancer stem cells in chronic myeloid leukemia.Curr Cancer Drug Targets. 2012; 12: 571-596Crossref Google Scholar including broad-spectrum cytotoxic agents against fast-proliferating cells and small-molecule inhibitors targeting specific signal transduction pathways, so called molecular therapies.4Ferrara F New agents for acute myeloid leukemia: is it time for targeted therapies?.Expert Opin Investig Drugs. 2012; 21: 179-189Crossref PubMed Scopus (0) Google Scholar The molecular pathogenesis of some leukemias, such as chronic myeloid leukemia (CML), is relatively clear; aberrant juxtaposition of BCR (breakpoint cluster region protein) and ABL1 (Abelson murine leukemia viral oncogene homolog 1) genes constitutively activates a tyrosine kinase (p210BCR-ABL), whose signaling initially leads to a chronic phase of myeloid cell expansion, while the expanded cells undergo differentiation in peripheral blood. A range of highly specific tyrosine kinase inhibitors (TKIs) has been introduced for clinical use over the last decade and significant improvements in patient survival have been achieved. For acute myeloid leukemia (AML), however, no new drugs have been introduced in recent years and clinical therapy has relied on “traditional” broad-spectrum cytotoxic drugs, where the leukemic cells display a differential sensitivity to drugs. The therapeutic index in this case is relatively small, and significant side effects at efficacious doses typically limit therapy at advanced disease.Leukemic cells generally respond well to drug therapy at the onset of treatment, but the drugs lose their effectiveness over a period of 6–12 months in a significant fraction of patients. It is now well recognized that the resistance to broad-spectrum drugs is inevitable, but recent evidence also indicated that even the most advanced molecular drugs can lose their efficacy.5Baccarani M Cortes J Pane F Niederwieser D Saglio G Apperley J et al.European LeukemiaNetChronic myeloid leukemia: an update of concepts and management recommendations of European LeukemiaNet.J Clin Oncol. 2009; 27: 6041-6051Crossref PubMed Scopus (811) Google Scholar In CML, development of resistance to current front-line therapy imatinib and failure to reach a complete cytogenetic response occurred in 24% of patients within 18 months.6Assouline S Lipton JH Monitoring response and resistance to treatment in chronic myeloid leukemia.Curr Oncol. 2011; 18: e71-e83Crossref PubMed Google Scholar,7O'Brien SG Guilhot F Larson RA Gathmann I Baccarani M Cervantes F et al.IRIS InvestigatorsImatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia.N Engl J Med. 2003; 348: 994-1004Crossref PubMed Scopus (2282) Google Scholar The inherent plasticity of the cells combined with diverse resistance mechanisms allow malignant cells to naturally adapt to drug assault. Additionally, the high relapse rate in leukemia patients has been attributed to existence of a rare population of leukemic stem cells (LSC) capable of evading drug therapies.8Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al.Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region.Nat Biotechnol. 2007; 25: 1315-1321Crossref PubMed Scopus (0) Google Scholar,9Mikkola HK Radu CG Witte ON Targeting leukemia stem cells.Nat Biotechnol. 2010; 28: 237-238Crossref PubMed Scopus (0) Google Scholar With better understanding of molecular changes in leukemic transformations, treatments that target tumor-specific changes are expected to lead to more effective therapies as the normal cells transform into malignant cells.To this end, a highly specific leukemia therapy can be developed by exploiting RNA interference (RNAi) to silence the aberrant protein(s) responsible for the disease.10Iorns E Lord CJ Turner N Ashworth A Utilizing RNA interference to enhance cancer drug discovery.Nat Rev Drug Discov. 2007; 6: 556-568Crossref PubMed Scopus (0) Google Scholar,11Rossbach M Small non-coding RNAs as novel therapeutics.Curr Mol Med. 2010; 10: 361-368Crossref PubMed Scopus (0) Google Scholar While current small molecular drugs rely on a specific binding mechanism, whether be an active enzyme site or DNA major/minor grooves, RNAi targets a particular mRNA for destruction (or translational blockage) by binding to specific regions in the mRNA. Unlike point mutations that can abolish drug activity, silencing aberrant proteins with RNAi is less prone to resistance development. The mechanism of action for RNAi reagents is similar to previously employed antisense oligonucleotides (AS-ODN) targeting mRNAs (Table 1), except that RNAi employs endogenous mRNA regulatory machinery to suppress protein production. Furthermore, RNAi can target aberrantly expressed isoform(s) of the protein (as in BCR-ABL fusion protein), unlike drugs that abolish activity of the target nonspecifically (as in both ABL and BCR-ABL proteins). RNAi for leukemia has reached clinical trials in two cases. In the NCT00257647, a viral vector, simian virus 40 (SV40), was utilized to deliver siRNA to CML patients against a fusion gene, but there are no published outcomes from this study. The other trial was a nonviral liposomal siRNA tested in one CML patient. A strategy to combine two or more drugs with nonoverlapping target resistance profiles could delay the emergence of drug resistance.12Sawyers CL Perspective: combined forces.Nature. 2013; 498: S7Crossref Scopus (0) Google Scholar However, new point mutations could still be expected to induce resistance to drug combinations,13Weisberg E Manley PW Cowan-Jacob SW Hochhaus A Griffin JD Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukaemia.Nat Rev Cancer. 2007; 7: 345-356Crossref PubMed Scopus (0) Google Scholar given the plasticity of LSC. FLT3 inhibitors (midostaurin, AC220 and sorafenib), for example, experience resistance development as a result of secondary FLT3-ITD mutations.14Moore AS Kearns PR Knapper S Pearson AD Zwaan CM Novel therapies for children with acute myeloid leukaemia.Leukemia. 2013; 27: 1451-1460Crossref PubMed Scopus (0) Google ScholarTable 1Different types of gene regulators used for leukemia therapyClass of CompoundsCharacteristicsSourceExamples in clinical leukemia therapyAntisense oligonucleotidesDouble-stranded DNA or modified formSyntheticGTI-2040 (ribonucleotide reductase), SPC2996 (Bcl-2), LY2181308 (survivin)Short interfering RNADouble-stranded, base-matched RNASyntheticBCR-ABL siRNAShort hairpin RNADouble-stranded, base-mismatched RNAIn situ expressedNot availableWhile AS-ODNs have reached clinical testing, only one siRNA, and no shRNA or microRNA were tested in clinics for leukemia therapy. Open table in a new tab The current review provides a comprehensive summary of RNAi efforts for leukemia therapy. We focus our analysis on myeloid leukemias, specifically AML and CML, where RNAi effort is mostly concentrated (but also provide information on other leukemias as appropriate). RNAi is a therapeutic option for all leukemias but we want to explore the critical issues in-depth that should be applicable to all leukemias (not just myeloid leukemias). We review the important aspects involved in utilization of RNAi reagents, with a particular focus on siRNA since it is likely to reach clinical testing ahead of other related reagents. Delivery of RNAi agents with nonviral carriers and factors affecting therapeutic efficacy have been emphasized. Where appropriate, experience with other types of RNAi reagents is summarized to generate a better sense of possible future progress. Finally, we provide the authors perspective on the future of RNAi in leukemic diseases, and identify hurdles and solutions to clinical deployment of RNAi technology.Technology of Nonviral RNAi DeliveryThe endogenous RNAi mechanism for post-transcriptional gene silencing is triggered by transcription of long pieces of double-stranded RNA (dsRNA) that are subsequently cleaved into smaller (21–23 nucleotides) microRNAs by Dicer.15De Paula D Bentley MV Mahato RI Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting.RNA. 2007; 13: 431-456Crossref PubMed Scopus (159) Google Scholar For a pharmacological RNAi intervention, a plasmid encoding for short hairpin RNA (shRNA) or a double-stranded siRNA, to bypass the shRNA transcription and processing steps, have been employed.16Whitehead KA Langer R Anderson DG Knocking down barriers: advances in siRNA delivery.Nat Rev Drug Discov. 2009; 8: 129-138Crossref PubMed Scopus (0) Google Scholar,17Guo P The emerging field of RNA nanotechnology.Nat Nanotechnol. 2010; 5: 833-842Crossref PubMed Scopus (0) Google Scholar The use of siRNA is more practical in hard-to-transfect primary cells and, in addition, it represents a more physiological mechanism to regulate gene expression as compared to AS-ODN18Fulda S Inhibitor of apoptosis proteins in pediatric leukemia: molecular pathways and novel approaches to therapy.Front Oncol. 2014; 4: 3Crossref Scopus (0) Google Scholar (Table 1). The siRNA is incorporated into the RNA-induced silencing complex (RISC), where Argonaute proteins cleave the sense strand of siRNA for release from the RISC. The activated RISC, which contains the antisense strand of siRNA, selectively seeks out and cleaves or represses the complementary mRNA.15De Paula D Bentley MV Mahato RI Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting.RNA. 2007; 13: 431-456Crossref PubMed Scopus (159) Google Scholar,16Whitehead KA Langer R Anderson DG Knocking down barriers: advances in siRNA delivery.Nat Rev Drug Discov. 2009; 8: 129-138Crossref PubMed Scopus (0) Google Scholar,19Yoda M Kawamata T Paroo Z Ye X Iwasaki S Liu Q et al.ATP-dependent human RISC assembly pathways.Nat Struct Mol Biol. 2010; 17: 17-23Crossref PubMed Scopus (0) Google Scholar While the activated RISC complex can move on to cleave additional mRNAs, it also gets diluted during cell division,15De Paula D Bentley MV Mahato RI Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting.RNA. 2007; 13: 431-456Crossref PubMed Scopus (159) Google Scholar so that repeated siRNA administration may be necessary to achieve a persistent effect. The large, hydrophilic, and anionic siRNA cannot cross plasma membrane and an effective carrier is needed to enable internalization and protection from almost immediate degradation by serum nucleases (Figure 1). Electroporation is a common method to deliver siRNA in culture by creating pores in cell membrane. While helpful to implement RNAi in culture,20Gioia R Leroy C Drullion C Lagarde V Etienne G Dulucq S et al.Quantitative phosphoproteomics revealed interplay between Syk and Lyn in the resistance to nilotinib in chronic myeloid leukemia cells.Blood. 2011; 118: 2211-2221Crossref PubMed Scopus (41) Google Scholar,21Kosova B Tezcanli B Ekiz HA Cakir Z Selvi N Dalmizrak A et al.Suppression of STAT5A increases chemotherapeutic sensitivity in imatinib-resistant and imatinib-sensitive K562 cells.Leuk Lymphoma. 2010; 51: 1895-1901Crossref Scopus (0) Google Scholar,22Tanaka N Wang YH Shiseki M Takanashi M Motoji T Inhibition of PRAME expression causes cell cycle arrest and apoptosis in leukemic cells.Leuk Res. 2011; 35: 1219-1225Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar such a method cannot be employed in vivo.23Merkerova M Bruchova H Kracmarova A Klamova H Brdicka R Bmi-1 over-expression plays a secondary role in chronic myeloid leukemia transformation.Leuk Lymphoma. 2007; 48: 793-801Crossref PubMed Scopus (0) Google Scholar,24Rangatia J Bonnet D Transient or long-term silencing of BCR-ABL alone induces cell cycle and proliferation arrest, apoptosis and differentiation.Leukemia. 2006; 20: 68-76Crossref PubMed Scopus (0) Google Scholar Viral vectors have been alternatively used both in in vitro and in vivo studies including the clinical trial NCT00257647.25Scherr M Chaturvedi A Battmer K Dallmann I Schultheis B Ganser A et al.Enhanced sensitivity to inhibition of SHP2, STAT5, and Gab2 expression in chronic myeloid leukemia (CML).Blood. 2006; 107: 3279-3287Crossref PubMed Scopus (0) Google Scholar,26Arthanari Y Pluen A Rajendran R Aojula H Demonacos C Delivery of therapeutic shRNA and siRNA by Tat fusion peptide targeting BCR-ABL fusion gene in Chronic Myeloid Leukemia cells.J Control Release. 2010; 145: 272-280Crossref PubMed Scopus (0) Google Scholar,27Schepers H van Gosliga D Wierenga AT Eggen BJ Schuringa JJ Vellenga E STAT5 is required for long-term maintenance of normal and leukemic human stem/progenitor cells.Blood. 2007; 110: 2880-2888Crossref PubMed Scopus (61) Google Scholar,28Tsai BY Suen JL Chiang BL Lentiviral-mediated Foxp3 RNAi suppresses tumor growth of regulatory T cell-like leukemia in a murine tumor model.Gene Ther. 2010; 17: 972-979Crossref Scopus (0) Google Scholar Although viral vectors are a prospective pursuit for leukemia, they present a significant safety risk due to their ability to integrate into a host's genome and/or cause significant immune responses,26Arthanari Y Pluen A Rajendran R Aojula H Demonacos C Delivery of therapeutic shRNA and siRNA by Tat fusion peptide targeting BCR-ABL fusion gene in Chronic Myeloid Leukemia cells.J Control Release. 2010; 145: 272-280Crossref PubMed Scopus (0) Google Scholar,29Mintzer MA Simanek EE Nonviral vectors for gene delivery.Chem Rev. 2009; 109: 259-302Crossref PubMed Scopus (0) Google Scholar and will not be further addressed in this review. Cationic biomolecules are safer for clinical deployment; they are capable of complexing and condensing anionic siRNA into spherical, stable nanoparticles (NPs) suitable for cellular uptake. Similar delivery systems can be employed for siRNA and AS-ODN since the molecular composition of siRNA is similar to AS-ODN.Functional carriers for RNAi agentsCarriers specifically explored for siRNA delivery in leukemic cells include cationic lipids, oligomers of cationic amino acids and other moieties, cationic polymers and various nano-structured materials (Table 2). Once the siRNA reaches the leukemic cell, it must gain entry through the cellular membrane, escape endosomes (if so entrapped) and get effectively released into the cytoplasm. The binding and engulfment of siRNA NPs at the plasma membrane require effective interactions to overcome the thermodynamics barriers to membrane poration.30Nel AE Mädler L Velegol D Xia T Hoek EM Somasundaran P et al.Understanding biophysicochemical interactions at the nano-bio interface.Nat Mater. 2009; 8: 543-557Crossref PubMed Scopus (2450) Google Scholar The lipid composition of the membrane as well as its dynamic nature influences internalization and may contribute to the difference in silencing among different cell types.31Janmey PA Kinnunen PK Biophysical properties of lipids and dynamic membranes.Trends Cell Biol. 2006; 16: 538-546Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar,32Lodish H Berk A Kaiser CA Krieger M Bretscher A Molecular Cell Biology. W H Freeman & Company, New York2007: 1Google Scholar The highly dynamic lipid rafts33Brown DA London E Structure and function of sphingolipid- and cholesterol-rich membrane rafts.J Biol Chem. 2000; 275: 17221-17224Crossref PubMed Scopus (1761) Google Scholar,34van Blitterswijk WJ Structural basis and physiological control of membrane fluidity in normal and tumor cells.Subcell Biochem. 1988; 13: 393-413Crossref PubMed Google Scholar may further “nucleate” interactions with siRNA NPs, leading to different type of affinities along the membrane.30Nel AE Mädler L Velegol D Xia T Hoek EM Somasundaran P et al.Understanding biophysicochemical interactions at the nano-bio interface.Nat Mater. 2009; 8: 543-557Crossref PubMed Scopus (2450) Google Scholar Creating cationic NPs capable of interacting with surface proteoglycans has been one approach to enhance siRNA uptake. Cationic single wall carbon nanotubes, for example, were used to silence cell-cycle regulator cyclin A2 in CML K562 cells;35Wang X Ren J Qu X Targeted RNA interference of cyclin A2 mediated by functionalized single-walled carbon nanotubes induces proliferation arrest and apoptosis in chronic myelogenous leukemia K562 cells.ChemMedChem. 2008; 3: 940-945Crossref PubMed Scopus (0) Google Scholar a significant (∼70%) reduction of cell numbers was obtained as a result of enhanced apoptosis. When cationic carriers are utilized for delivery, increasing the carrier:siRNA ratio (often referred as the N/P -amine/phosphate- ratio) often improves delivery as a result of increased charge of the complex.36Landry B Aliabadi HM Samuel A Gül-Uludag H Jiang X Kutsch O et al.Effective non-viral delivery of siRNA to acute myeloid leukemia cells with lipid-substituted polyethylenimines.PLoS ONE. 2012; 7: e44197Crossref PubMed Scopus (0) Google Scholar,37Omedes Pujol M Coleman DJ Allen CD Heidenreich O Fulton DA Determination of key structure-activity relationships in siRNA delivery with a mixed micelle system.J Control Release. 2013; 172: 939-945Crossref Scopus (0) Google Scholar The cellular uptake of siRNA (binding and internalization) is generally observed to occur within a few hours for both targeted and untargeted carriers, and less (e.g., ∼1 hour) for liposomes in AML cells38Rothdiener M Müller D Castro PG Scholz A Schwemmlein M Fey G et al.Targeted delivery of SiRNA to CD33-positive tumor cells with liposomal carrier systems.J Control Release. 2010; 144: 251-258Crossref PubMed Scopus (38) Google Scholar and albumin-coated cell-penetrating peptides (CPPs) in ATLL cells.39Hou KK Pan H Ratner L Schlesinger PH Wickline SA Mechanisms of nanoparticle-mediated siRNA transfection by melittin-derived peptides.ACS Nano. 2013; 7: 8605-8615Crossref PubMed Scopus (0) Google Scholar Interestingly, a high peak delivery (96%) was achieved with a targeted peptide system at ∼2 hours with a rapid decline thereafter.40Lee YK Kim KS Kim JS Baek JE Park SI Jeong HY et al.Leukemia-specific siRNA delivery by immunonanoplexes consisting of anti-JL1 minibody conjugated to oligo-9 Arg-peptides.Mol Cells. 2010; 29: 457-462Crossref PubMed Scopus (0) Google Scholar siRNA silencing was not demonstrated with this system and the reason of the rapid decline was not discussed, but could indicate siRNA release (affecting measurable fluorescence levels) or perhaps even exocytosis. siRNA delivery studies, performed with lipid- polyethylenimine (PEI) carrier libraries in CML cells and breast cancer cells confirmed the lower delivery percentage in CML cells. These results initiated further formulation alterations to achieve more comparable delivery in the CML cells.41Valencia-Serna J Gul-Uludag H Mahdipoor P Jiang X Uludag H Investigating siRNA delivery to chronic myeloid leukemia K562 cells with lipophilic polymers for therapeutic BCR-ABL down-regulation.J Control Release. 2013; 172: 495-503Crossref PubMed Scopus (0) Google ScholarTable 2Nonviral, noncommercial carriers developed for siRNA-based therapy of leukemiaDeliveryaDelivery reported if measured by fluorescent siRNA.SilencingbSilencing of a target if reported with indicated siRNA concentration (nmol/l). (nmol/l)Therapeutic effectcTherapeutic effect of silencing a protein target, if reported with indicated siRNA concentration (nmol/l). “V” indicates study was performed and significant results obtained. “X' indicates study was not performed or significant results were not obtained. In all cases, a “V” is only indicated when the studies were performed in leukemic cells. In some cases, delivery, silencing and/or therapeutic effects were demonstrated in nonleukemic cells, the results of which are not shown. “!” Indicates study was done in nonleukemic cells. “λ” Indicates testing was also done in other cell types. (nmol/l)Ref.siRNA carrierCarrier design rationalesiRNA targets (cell)In vitroIn vivoIn vitroIn vivoIn vitroIn vivoMultiple leukemia types45Lipid NP; Cationic lipids (with alkylated DMA) + neutral lipids + PEG coatingAlkylated DMA key for improved transfection (increased particle order and stability). Protamine, HA, peptides (PPAA and INF7) enhanced transfection.KIF11, FLT3 (AML (THP-1, KG-1, Molm13, Mv4-11, HEL), CML (K562), Molm13 in vivo.)XX√ (10-500)√!√ (10-30)X73,120.Modified siRNA; TLR9 antagonist CpGNot a carrier. Targeted delivery for siRNA. Does not protect against serum nucleasesSTAT3 (AML (MV4-11, patient), MM (KMS-11, patient), TLR9+ hematopoietic cells, human PB blood cells (monocytes, T cells, NK cells, B cells, mDCs, pDCs)^√√√ (500 in MM)√√!√42,43Peptide; CPP PepFect6Characterized and tested amphipathic and arginine-rich CPPs for siRNA silencing. Amphipathic PepFect6 was the most promising CPP (Comprised of stearyl-TP10 peptide with trifluoromethylquinoline moieties for endosomal escape, effective with serum proteins). Electrostatically formed NPLuciferase (reporter gene) (AML (SKNO-1)); HPRT1; (CML (K562) and ALL (Jurkat)) Λ√; XX; X√ (50-200); V (12.5-100)X; ✓!X; XX; X44Fusion protein; PTD- DRBDDRBD for binding to siRNA, PTD for cellular delivery. Developed for delivery to primary cell and thus also tested in other cell types/animal models. DRBD avidity to siRNA mediated NP formation.GFP (THP-1 differentiated to macrophages?) ALL (Jurka)^XX√ (100-400)XXX46Lipid NP; Transferrin ligand + cationic lipid DODMAMicrofluidic formation for controlled mixing parameters during self-assembly.RRM2 (AML (MV4-11) and CML (κ562))λ√√√ (100-500)√√ (100-1,000)XAML36,118 119Polymeric complex; Lipids for cell interactions. Caprylic or linoleic acid Low MW PEI for decreased substituted on 2kDa toxicity while maintaining PEI beneficial properties of PEI.GFP (reporter gene), CXCR4, SDF-1, CD44 (THP-1, KG-1, KG-1a, patient)√X√ (25-100)X√ (25-100)X49Chitosan NP; ChitosanChitosan is biocompatible, cationic, and adhesive. Electrostatically formed NPVEGF, FLT1 (U937)XX√; UnknownX√; UnknownX37Micelle; Amphiphilic diblock copolymers (4.5 kDa PCL with 15.5 kDa PDMAEMA or 5 kDa PEG).PDMAEMA provides charge for siRNA binding and buffering for endosomal rupture. PEG provides colloidal stability/RES protection. Two polymers allow for cell type optimization through cationic charge and resulting toxicity.Luciferase (reporter gene), RUNX1/ETO (SKNO-1)^√X√ (500)XXX38Liposome; Anti-CD33 Ab + EPC/chol/mPEG 2000-DSPE w/wo PEI 25 kDa coreCD33 for cell targeting; PEI (electrostatically binding to siRNA) increased liposome encapsulation of siRNA however PEI encapsulation did not improve silencing results.AML1/MTG8 fusion protein (SKNO-1, Kasumi-1)√X√ (600-2,500)X√ (30-125)XCML41Polymeric complex; Palmitic acid substituted on 1.2 kDa PEILipid for enhanced cell interactions. Low MW PEI for decrease toxicity. Electrostatically formed complexes.GFP (reporter gene), BCR-ABL (K562)√X√(36-100)X√ (50-100)X50Chitosan NP; ChitosanChitosan for siRNA delivery Tested in multiple cell types including CML. Electrostatically formed NP.BCR-ABL (K562)^XX√XXX26Peptide; CPP (HIV-Tat (49-57)) + membrane lytic peptide (LK15)Peptides for cytoplasm delivery and endosomal escape. Shows studies with shRNA and siRNA. Electrostatically formed NP.BCR-ABL (K562)√X√ (24-729 Est.)X√ (1, 428-2, 142, Est.)X35, 147Carbon Nanotubes; Ammonium functionalized SWNTSWNT provides high siRNA loading. Ammonium provides positive charge to bind siRNA. Electrostatically formed.Cyclin A2XX√ (25)X√ (25)X47Liposome; Transferrin- PEG2000- DSPE + Chol/DSPC/ DODAP/C16 mPEG 2000 CeramideDODAP (+ve at pH 4/neutral at physiological pH) and optimized buffer concentration provides high siRNA encapsulationBCR-ABL (K562 and LAMA-84)√X√ (500-2, 000)X√ (500-2, 000)XALL and ATLL40Peptide; Minibody (anti-JL1) conjugated to oligo-9-Arg- peptide CPPSpecific mini-body against JL1 (specific to leukemic cells and not mature hematopoietic cell). CPP for internalization. Electrostatically formedNo target (FITC scrambled siRNA) (CCRF-CEM, Jurkat)√√XXXX51NP; Chitosan + TPPChitosan is biocompatible and adhesive. (Adhesion properties may promote tumor survival). Electrostatically formed NP.Hsp70 (Jurkat)XX√ 50X√ (50)X39NP; Albumin coated CPP complexAlbumin coating for stabilization and prevention of flocculation. Melittin derived P5RHH peptide for endosomal escape. Electrostatically formed NP.p65 and p100/52 (NfκB) (F8)√√√ (50-200 Est.)X√ (50-200 Est.)XVarious carrier formulations were categorized based on the type of leukemia they were tested in.AML, acute myeloid leukemia; CPP, cell penetrating peptide; C16 mPEG 2000 Ceramide, N-palmitoyl-sphingosine- 1-(succinyl(methoxypolyethylene glycol) 2000); Chol, cholesterol; CML, chronic myeloid leukemia; DC-Chol, 3b-(N-(N,N-dimethylaminoethane)-carbamoyl) hydrochloride; DSPC, 1,2-distearoyl-sn- glycero-3-phosphatidylcholine; DODAP, 1,2-dioleoyl-3-dimethylammonium-propane; DODMA, dioleyloxy-N,N-dimethyl-3-aminopropane; Egg PC, Egg phosphatidylcholine; HA, hyaluronic acid; MW, molecular weight; NP, nanoparticle; PEG, polyethylene glycol; PCL, polycaprolactone; PDMAEMA, poly((dimethylamino) ethylene methacrylate); PEI, polyethylenimine; PPAA, poly(propylacrylic acid) peptide; RES, reticuloendothelial system; TPP, tripolyphosphate; Trf, Transferrin. “Est” indicates concentration was estimated from the data provided.a Delivery reported if measured by fluorescent siRNA.b Silencing of a target if reported with indicated siRNA concentration (nmol/l).c Therapeutic effect of silencing a protein target, if reported with indicated siRNA concentration (nmol/l). “V” indicates study was performed and significant results obtained. “X' indicates study was not performed or significant results were not obtained. In all cases, a “V” is only indicated when the studies were performed in leukemic cells. In some cases, delivery, silencing and/or therapeutic effects were demonstrated in nonleukemic cells, the results of which are not shown. “!” Indicates study was done in nonleukemic cells. “λ” Indicates testing was also done in other cell types. Open table in a new tab Cationic CPPs26Arthanari Y Pluen A Rajendran R Aoju
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