Ly James Lee

Targeted Delivery of microRNA-29b by Transferrin-Conjugated Anionic Lipopolyplex Nanoparticles: A Novel Therapeutic Strategy in Acute Myeloid Leukemia

Purpose: miR-29b directly or indirectly targets genes involved in acute myeloid leukemia (AML), namely, DNMTs, CDK6, SP1, KIT, and FLT3. Higher miR-29b pretreatment expression is associated with improved response to decitabine and better outcome in AML. Thus, designing a strategy to increase miR-29b levels in AML blasts may be of therapeutic value. However, free synthetic miRs are easily degraded in bio-fluids and have limited cellular uptake. To overcome these limitations, we developed a novel transferrin-conjugated nanoparticle delivery system for synthetic miR-29b (Tf-NP-miR-29b). Experimental Design: Delivery efficiency was investigated by flow cytometry, confocal microscopy, and quantitative PCR. The expression of miR-29b targets was measured by immunoblotting. The antileukemic activity of Tf-NP-miR-29b was evaluated by measuring cell proliferation and colony formation ability and in a leukemia mouse model. Results: Tf-NP-miR-29b treatment resulted in more than 200-fold increase of mature miR-29b compared with free miR-29b and was approximately twice as efficient as treatment with non-transferrin–conjugated NP-miR-29b. Tf-NP-miR-29b treatment significantly downregulated DNMTs, CDK6, SP1, KIT, and FLT3 and decreased AML cell growth by 30% to 50% and impaired colony formation by approximately 50%. Mice engrafted with AML cells and then treated with Tf-NP-miR-29b had significantly longer survival compared with Tf-NP-scramble (P = 0.015) or free miR-29b (P = 0.003). Furthermore, priming AML cell with Tf-NP-miR-29b before treatment with decitabine resulted in marked decrease in cell viability in vitro and showed improved antileukemic activity compared with decitabine alone (P = 0.001) in vivo. Conclusions: Tf-NP effectively delivered functional miR-29b, resulting in target downregulation and antileukemic activity and warrants further investigation as a novel therapeutic approach in AML. Clin Cancer Res; 19(9); 2355–67. ©2013 AACR.

Lactosylated Gramicidin-based lipid nanoparticles (Lac-GLN) for targeted delivery of anti-miR-155 to hepatocellular carcinoma

Lactosylated gramicidin-containing lipid nanoparticles (Lac-GLN) were developed for delivery of anti-microRNA-155 (anti-miR-155) to hepatocellular carcinoma (HCC) cells. MiR-155 is an oncomiR frequently elevated in HCC. The Lac-GLN formulation contained N-lactobionyl-dioleoyl phosphatidylethanolamine (Lac-DOPE), a ligand for the asialoglycoprotein receptor (ASGR), and an antibiotic peptide gramicidin A. The nanoparticles exhibited a mean particle diameter of 73 nm, zeta potential of +3.5mV, anti-miR encapsulation efficiency of 88%, and excellent colloidal stability at 4°C. Lac-GLN effectively delivered anti-miR-155 to HCC cells with a 16.1- and 4.1-fold up-regulation of miR-155 targets C/EBPβ and FOXP3 genes, respectively, and exhibited significant greater efficiency over Lipofectamine 2000. In mice, intravenous injection of Lac-GLN containing Cy3-anti-miR-155 led to preferential accumulation of the anti-miR-155 in hepatocytes. Intravenous administration of 1.5 mg/kg anti-miR-155 loaded Lac-GLN resulted in up-regulation of C/EBPβ and FOXP3 by 6.9- and 2.2-fold, respectively. These results suggest potential application of Lac-GLN as a liver-specific delivery vehicle for anti-miR therapy.

Lenalidomide-mediated enhanced translation of C/EBPα-p30 protein up-regulates expression of the antileukemic microRNA-181a in acute myeloid leukemia

Recently, we showed that increased miR-181a expression was associated with improved outcomes in cytogenetically normal acute myeloid leukemia (CN-AML). Interestingly, miR-181a expression was increased in CN-AML patients harboring CEBPA mutations, which are usually biallelic and associate with better prognosis. CEBPA encodes the C/EBPα transcription factor. We demonstrate here that the presence of N-terminal CEBPA mutations and miR-181a expression are linked. Indeed, the truncated C/EBPα-p30 isoform, which is produced from the N-terminal mutant CEBPA gene or from the differential translation of wild-type CEBPA mRNA and is commonly believed to have no transactivation activity, binds to the miR-181a-1 promoter and up-regulates the microRNA expression. Furthermore, we show that lenalidomide, a drug approved for myelodysplastic syndromes and multiple myeloma, enhances translation of the C/EBPα-p30 isoform, resulting in higher miR-181a levels. In xenograft mouse models, ectopic miR-181a expression inhibits tumor growth. Similarly, lenalidomide exhibits antitumorigenic activity paralleled by increased miR-181a expression. This regulatory pathway may explain an increased sensitivity to apoptosis-inducing chemotherapy in subsets of AML patients. Altogether, our data provide a potential explanation for the improved clinical outcomes observed in CEBPA-mutated CN-AML patients, and suggest that lenalidomide treatment enhancing the C/EBPα-p30 protein levels and in turn miR-181a may sensitize AML blasts to chemotherapy.

Adjuvanted poly(lactic-co-glycolic) acid nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine elicits cross-protective immune response in pigs

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is an economically devastating disease, causing daily losses of approximately

Pharmacological targeting of miR-155 via the NEDD8-activating enzyme inhibitor MLN4924 (Pevonedistat) in FLT3-ITD acute myeloid leukemia

3 million to the US pork industry. Current vaccines have failed to completely prevent PRRS outbreaks. Recently, we have shown that poly(lactic-co-glycolic) acid (PLGA) nanoparticle-entrapped inactivated PRRSV vaccine (NP-KAg) induces a cross-protective immune response in pigs. To further improve its cross-protective efficacy, the NP-KAg vaccine formulation was slightly modified, and pigs were coadministered the vaccine twice intranasally with a potent adjuvant: Mycobacterium tuberculosis whole-cell lysate. In vaccinated virulent heterologous PRRSV-challenged pigs, the immune correlates in the blood were as follows: 1) enhanced PRRSV-specific antibody response with enhanced avidity of both immunoglobulin (Ig)-G and IgA isotypes, associated with augmented virus-neutralizing antibody titers; 2) comparable and increased levels of virus-specific IgG1 and IgG2 antibody subtypes and production of high levels of both T-helper (Th)-1 and Th2 cytokines, indicative of a balanced Th1–Th2 response; 3) suppressed immunosuppressive cytokine response; 4) increased frequency of interferon-γ+ lymphocyte subsets and expanded population of antigen-presenting cells; and most importantly 5) complete clearance of detectable replicating challenged heterologous PRRSV and close to threefold reduction in viral ribonucleic acid load detected in the blood. In conclusion, intranasal delivery of adjuvanted NP-KAg vaccine formulation to growing pigs elicited a broadly cross-protective immune response, showing the potential of this innovative vaccination strategy to prevent PRRS outbreaks in pigs. A similar approach to control other respiratory diseases in food animals and humans appears to be feasible.

Targeting Leukemia Stem Cells in vivo with AntagomiR-126 Nanoparticles in Acute Myeloid Leukemia

High levels of microRNA-155 (miR-155) are associated with poor outcome in acute myeloid leukemia (AML). In AML, miR-155 is regulated by NF-κB, the activity of which is, in part, controlled by the NEDD8-dependent ubiquitin ligases. We demonstrate that MLN4924, an inhibitor of NEDD8-activating enzyme presently being evaluated in clinical trials, decreases binding of NF-κB to the miR-155 promoter and downregulates miR-155 in AML cells. This results in the upregulation of the miR-155 targets SHIP1, an inhibitor of the PI3K/Akt pathway, and PU.1, a transcription factor important for myeloid differentiation, leading to monocytic differentiation and apoptosis. Consistent with these results, overexpression of miR-155 diminishes MLN4924-induced antileukemic effects. In vivo, MLN4924 reduces miR-155 expression and prolongs the survival of mice engrafted with leukemic cells. Our study demonstrates the potential of miR-155 as a novel therapeutic target in AML via pharmacologic interference with NF-κB-dependent regulatory mechanisms. We show the targeting of this oncogenic microRNA with MLN4924, a compound presently being evaluated in clinical trials in AML. As high miR-155 levels have been consistently associated with aggressive clinical phenotypes, our work opens new avenues for microRNA-targeting therapeutic approaches to leukemia and cancer patients.

Milatuzumab-Conjugated Liposomes as Targeted Dexamethasone Carriers for Therapeutic Delivery in CD74+ B-cell Malignancies

Current treatments for acute myeloid leukemia (AML) are designed to target rapidly dividing blast populations with limited success in eradicating the functionally distinct leukemia stem cell (LSC) population, which is postulated to be responsible for disease resistance and relapse. We have previously reported high miR-126 expression levels to be associated with a LSC-gene expression profile. Therefore, we hypothesized that miR-126 contributes to ‘stemness’ and is a viable target for eliminating the LSC in AML. Here we first validate the clinical relevance of miR-126 expression in AML by showing that higher expression of this microRNA (miR) is associated with worse outcome in a large cohort of older (⩾60 years) cytogenetically normal AML patients treated with conventional chemotherapy. We then show that miR-126 overexpression characterizes AML LSC-enriched cell subpopulations and contributes to LSC long-term maintenance and self-renewal. Finally, we demonstrate the feasibility of therapeutic targeting of miR-126 in LSCs with novel targeting nanoparticles containing antagomiR-126 resulting in in vivo reduction of LSCs likely by depletion of the quiescent cell subpopulation. Our findings suggest that by targeting a single miR, that is, miR-126, it is possible to interfere with LSC activity, thereby opening potentially novel therapeutic approaches to treat AML patients.

Tumor antigen ROR1 targeted drug delivery mediated selective leukemic but not normal B-cell cytotoxicity in chronic lymphocytic leukemia.

Purpose: Corticosteroids are widely used for the treatment of B-cell malignancies, including non–Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia; however, this class of drug is associated with undesirable off-target effects. Herein, we developed novel milatuzumab-conjugated liposomes as a targeted dexamethasone carrier for therapeutic delivery in CD74+ B-cell malignancies and explored its effect against the disease. Experimental Design: The targeting efficiency of milatuzumab-targeted liposomes to CD74+ cells was evaluated in vitro. The effect of CD74-targeted liposomal dexamethasone was compared with free dexamethasone in primary CLL cells and cell lines in vitro. The therapeutic efficacy of CD74-targeted liposomal dexamethasone was evaluated in a Raji-severe combined immunodeficient (SCID) xenograft model in vivo. Results: Milatuzumab-targeted liposomes promoted selective incorporation of carrier molecules into transformed CD74-positive B cells as compared with CD74-negative T-cells. The CD74-dexamethasone–targeted liposomes (CD74-IL-DEX) promoted and increased killing in CD74-positive tumor cells and primary CLL cells. Furthermore, the targeted drug liposomes showed enhanced therapeutic efficacy against a CD74-positive B-cell model as compared with free, or non-targeted, liposomal dexamethasone in SCID mice engrafted with Raji cells in vivo. Conclusions: These studies provide evidence and support for a potential use of CD74-targeted liposomal dexamethasone as a new therapy for B-cell malignancies. Clin Cancer Res; 19(2); 347–56. ©2012 AACR.

Micro-/nano-electroporation for active gene delivery.

Selective cytotoxicity to cancer cells without compromising their normal counterparts pose a huge challenge for traditional drug design. Here we developed a tumor antigen-targeted delivery of immunonanoparticle carrying a novel non-immunosuppressive FTY720 derivative OSU-2S with potent cytotoxicity against leukemic B cells. OSU-2S induces activation of protein phosphatase 2A (PP2A), phosphorylation and nuclear translocation of SHP1S591 and deregulation of multiple cellular processes in chronic lymphocytic leukemia (CLL) resulting in potent cytotoxicity. To preclude OSU-2S-mediated effects on these ubiquitous phosphatases in unintended cells and avoid potential adverse effects, we developed an OSU-2S-targeted delivery of immunonanoparticles (2A2-OSU-2S-ILP), that mediated selective cytotoxicity of CLL but not normal B cells through targeting receptor tyrosine kinase ROR1 expressed in leukemic but not normal B cells. Developing a novel spontaneous CLL mouse model expressing human ROR1 (hROR1) in all leukemic B cells, we demonstrate the therapeutic benefit of enhanced survival with 2A2-OSU-2S-ILP in vivo. The newly developed non-immunosuppressive OSU-2S, its delivery using human CLL directed immunonanoparticles and the novel transgenic (Tg) mouse model of CLL that expresses hROR1 exclusively in leukemic B cell surface are highly innovative and can be applied to CLL and other ROR1+ malignancies including mantle cell lymphoma and acute lymphoblastic leukemia.

Micro-/nanoscale electroporation

Gene delivery, a process of introducing foreign functional nucleic acids into target cells, has proven to be a very promising tool for inducing specific gene expression in host cells. Many different technologies have been developed for efficient gene delivery. Among them, electroporation has been adopted in gene delivery for decades, and it is currently widely used for transfection of different types of cells. Despite of the success achieved by bulk electroporation (BEP) for gene delivery in vitro and in vivo, it has significant drawbacks such as unstable transfection efficacy and low cell viability. In recent years, there is an emerging interest in understanding how individual cell accepts and responds to exogenous gene materials using single cell based micro-/nano-electroporation (MEP/NEP) technologies. In this review, the authors provide an overview of the recent development of MEP/NEP and their advantages in gene delivery. Additionally, the future perspectives of gene delivery with the application of electroporation are discussed.