The Challenges and Strategies of Antisense Oligonucleotide Drug Delivery

Antisense oligonucleotides (ASOs) are used to selectively inhibit the translation of disease-associated genes via Ribonuclease H (RNaseH)-mediated cleavage or steric hindrance. They are being developed as a novel and promising class of drugs targeting a wide range of diseases. Despite the great potential and numerous ASO drugs in preclinical research and clinical trials, there are many limitations to this technology. In this review we will focus on the challenges of ASO delivery and the strategies adopted to improve their stability in the bloodstream, delivery to target sites, and cellular uptake. Focusing on liposomal delivery, we will specifically describe liposome-incorporated growth factor receptor-bound protein-2 (Grb2) antisense oligodeoxynucleotide BP1001. BP1001 is unique because it is uncharged and is essentially non-toxic, as demonstrated in preclinical and clinical studies. Additionally, its enhanced biodistribution makes it an attractive therapeutic modality for hematologic malignancies as well as solid tumors. A detailed understanding of the obstacles that ASOs face prior to reaching their targets and continued advances in methods to overcome them will allow us to harness ASOs’ full potential in precision medicine.

Targeting transforming growth factor-β2 by antisense oligodeoxynucleotide accelerates T cell-mediated tumor rejection in a humanized mouse model of triple-negative breast cancer

Background: Transforming growth factor (TGF-β) pathway mediates suppression of anti-tumor immunity, and is associated with poor prognosis in triple-negative breast cancer (TNBC). Methods: In this study, we generated a humanized animal model by transplanting human peripheral blood mononuclear cells into immunodeficient mice followed by inoculation of MDA-MB-231 cells, and subsequently analyzed the role of TGF-β2 in the interaction between human T cells and human tumor cells. Results: Following reconstitution of the human immune system, inhibition of TGF-β signaling by TGF-β2 antisense oligodeoxynucleotide (TASO) resulted in accelerated tumor growth inhibition. TGF-β2 inhibition also resulted in downregulation of peripheral Foxp3+ regulatory T cells (Treg), whereas no effect was seen in the expression of CD8+ cytotoxic T cells. Analysis of the TASO-treated mice serum revealed elevated levels of human IFN-γ and reduced levels of human IL-10 and TGF-β2. Moreover, TGF-β2 inhibition resulted in increased CD8+ T cell infiltration, whereas the reduced infiltration of Tregs into the tumor partly resulted from decreased expression of CCL22. Decreased intra-tumoral Tregs facilitated the activation of cytotoxic T cells, associated with increased granzyme B expression. Conclusion: These results indicate that TASO potentiated T-cell mediated antitumor immunity, and it is proposed that TGF-β2 may be a promising target in the immunotherapeutic strategy of TNBC.

Targeting MiR-126 Blocks Progression of Myelodysplastic Syndrome and Transformation into Acute Myeloid Leukemia

Clonal-analysis studies have demonstrated that MDS is possibly derived from clonal hematopoietic stem cells (HSC) or early myeloid progenitors. These aberrant hematopoietic stem and progenitor cells (HSPCs) are resistant to therapy and expand during relapse. Understanding mechanisms contributing to their homeostasis is critical. MiR-126-3p (miR-126) is highly expressed in myeloid leukemia stem cell and restrains cell-cycle progression (Nature Medicine, 2018). Additionally, higher miR-126 levels reportedly correlate with poor outcome in MDS patients (pts; Korean J Intern Med, 2019). Thus, we postulated that miR-126 may play a relevant role in MDS pathogenesis. Accordingly, we found miR-126 levels were negatively correlated with overall survival [higher miR-126 (n=28) vs lower miR-126 (n=28) expression: 804 days vs undefined, p<0.05] and associated with leukemia transformation (transformation vs no transformation: 34.55±12.18 vs 13.37±2.89, p<0.05) in a cohort of consecutive MDS cases seen at our institution. Notably, miR-126 levels were higher in CD34+CD38- relative to CD34+CD38+ (0.32±0.03 vs 0.18±0.02, p<0.01) from same MDS pts. Similarly, when analyzed NUP19/HOXD13 (NHD13) mouse which recapitulates well human MDS (PANS, 2008), we found miR-126 levels were higher in the more primitive subset of Linage-c-kit+sca-1+ (LSK) cells relative to a more mature cells (Linage-c-kit+sca-1-, LK) (1.55±0.36 vs 0.13±0.04, p<0.01). Knockdown of miR-126 in a human MDS derived cell line (MDS-L) using short hairpin RNA (shmiR-126) increased cell cycling (G1: KD 52.4%±1.2% vs ctrl 63.9%±3.0%, p<0.05) and apoptosis (KD 18.8%±1.2% vs ctrl 2.0%±0.3%, p<0.001). We have recently developed a novel miR-126 inhibitor (miRsten) by linking a miR-126-specific antisense oligodeoxynucleotide (ODN) to a cytosine-guanine dinucleotide (CpG) ODN(Nature Medicine, 2018. We therefore tested the combination of miRsten with decitabine (DAC) on primary MDS CD34+ cells; cells exposed to miRsten were more sensitive to DAC treatment than controls cells (miRsten +DAC [combination]: 46.3%±3.2% vs DAC 30.5%±1.4%, p<0.05, n=4). MDS CD34+cells treated exvivo with the combination (comb) of miRsten and DAC for 72 hrs had reduced engraftment ability in NSGS mice compared with cells treated with either drug alone or vehicle at 12 wks post-BMT (miRsten 2.7%±0.3% vs vehicle 5.6%±0.3%, p<0.0001; comb 0.8%±0.2% vs vehicle 5.6%±0.3%, p<0.0001; comb 0.8%±0.2% vs miRsten 2.7%±0.3%, p<0.001; comb 0.8%±0.2% vs DAC 1.5%±0.2%, p<0.05, n=5). Dysplastic CD45.2+ BM cells from donor NHD13+ mice were transplanted into preconditioned congenic recipients expressing CD45.1. The transplanted animals were treated with vehicle, miRsten (10mg/kg/day), DAC (0.5mg/kg, 3 times/ week) or combination of miRsten and DAC for 3 wks (n=6). Combination treatment resulted in partial reversal of MDS-like disease, with a significant increase in hemoglobin, platelet, red blood cell and white blood cell. The combination also significantly reduced NHD13+ chimerism (miRsten 73.6%±2.9% vs vehicle 87.2%±2.3%, p<0.01; comb 50.2%±4.4% vs vehicle 87.2%±2.3%, p<0.001; comb 50.2%±4.4% vs miRsten 73.6%±2.9%, p<0.01; comb 50.2%±4.4% vs DAC 67.8%±4.1%, p<0.05, n=6). In secondary transplantation experiments, the mice receiving BM cells from comb-treated donors showed reduced NHD13+ CD45.2+ cell long-term engraftment compared with recipients of cells from donors treated with either drug alone or vehicle (miRsten 28.6%±5.1% vs vehicle 36.7%±7.1%, p<0.06; comb 9.1%±2.1% vs vehicle 36.7%±7.1%, p<0.0001; comb 9.1%±2.1% vs miRsten 28.6%±5.1%, p<0.01; comb 9.1%±2.1% vs DAC 23.3%±5.3%, p<0.05, n=7). We also monitored the leukemia transformation in a cohort of tertiary transplants. After donor cells robust engrafted, we treated the transplants as above four groups. Notably, leukemia transformation was delayed, evidenced by reduced WBC and blast counts as well as extended survival in recipients of cells from donors treated with comb (median survival: miRsten 102d vs vehicle 34d, p<0.0001; comb 188d vs vehicle 34d, p<0.0001; comb 188d vs miRsten 102d, p<0.0001; comb 188d vs DAC 67d, p<0.0001, n=10). Taken together, our results indicate that miR-126 targeting may disrupt MDS cell growth in vivo; mechanistic studies to explain how miR-126 contributes to MDS maintenance and optimize therapeutic targeting of this miR-126 with miRsten are ongoing. Disclosures Stein: Celgene: Speakers Bureau; Stemline: Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

Sirtuin 1 Regulates Mitochondrial Biogenesis and Provides an Endogenous Neuroprotective Mechanism Against Seizure-Induced Neuronal Cell Death in the Hippocampus Following Status Epilepticus

Status epilepticus may decrease mitochondrial biogenesis, resulting in neuronal cell death occurring in the hippocampus. Sirtuin 1 (SIRT1) functionally interacts with peroxisome proliferator-activated receptors and γ coactivator 1α (PGC-1α), which play a crucial role in the regulation of mitochondrial biogenesis. In Sprague-Dawley rats, kainic acid was microinjected unilaterally into the hippocampal CA3 subfield to induce bilateral seizure activity. SIRT1, PGC-1α, and other key proteins involving mitochondrial biogenesis and the amount of mitochondrial DNA were investigated. SIRT1 antisense oligodeoxynucleotide was used to evaluate the relationship between SIRT1 and mitochondrial biogenesis, as well as the mitochondrial function, oxidative stress, and neuronal cell survival. Increased SIRT1, PGC-1α, and mitochondrial biogenesis machinery were found in the hippocampus following experimental status epilepticus. Downregulation of SIRT1 decreased PGC-1α expression and mitochondrial biogenesis machinery, increased Complex I dysfunction, augmented the level of oxidized proteins, raised activated caspase-3 expression, and promoted neuronal cell damage in the hippocampus. The results suggest that the SIRT1 signaling pathway may play a pivotal role in mitochondrial biogenesis, and could be considered an endogenous neuroprotective mechanism counteracting seizure-induced neuronal cell damage following status epilepticus.

RP3V kisspeptin neurons mediate neuroprogesterone induction of the luteinizing hormone surge in female rat

ABSTRACTTo induce ovulation, neural circuits are sequentially activated by estradiol and progesterone. In female rodents, estradiol-induced neuroprogesterone, synthesized in astrocytes, is essential for the luteinizing hormone (LH) surge and subsequently, ovulation. However, the gonadotropin-releasing hormone (GnRH) neurons do not express the steroid receptors necessary for reproduction: progesterone receptors (PGR) or estrogen receptor-α (ERα). Steroid information is transduced by rostral periventricular (RP3V) kisspeptin neurons that express ERα and PGR and innervate GnRH neurons in the diagonal band of Broca (DBB) and the medial septum. In this study, we tested the hypothesis that estradiol induced neuroprogesterone needed for the LH surge is mediated by kisspeptin. Neuroprogesterone synthesis was inhibited with aminoglutethimide (AGT; s.c.) in 17β-estradiol benzoate (EB)-primed, ovariectomized (ovx) and adrenalectomized (adx) rats. Kisspeptin-10 (20 nmol/µl) was infused into the DBB, trunk blood was collected 53 hours post-EB injection, and serum LH levels were analyzed by ELISA. AGT inhibition of neuroprogesterone synthesis blocked the EB-induced LH surge. Subsequent treatment with either progesterone or DBB kisspeptin-10 infusion restored the LH surge. Kisspeptin restored the LH surge, which was blocked by DBB infusion of kisspeptin receptor (GPR54) antagonist (kisspeptin-234). Finally, knockdown of kisspeptin protein levels in the RP3V with kisspeptin antisense oligodeoxynucleotide (ODN) significantly lowered LH levels in EB-primed rats compared to scrambled ODN, demonstrating the importance of endogenous RP3V kisspeptin for the LH surge. These results support the hypothesis that neuroprogesterone induces both kisspeptin release from RP3V neurons impacting the LH surge.