RESUMO
Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here we isolated a high-affinity (HA) folate receptor beta (FRß)-specific single-chain variable fragment (2.48 nm KD) for optimization of FRß-redirected CAR T-cell therapy for AML. T cells stably expressing the HA-FRß CAR exhibited greatly enhanced antitumor activity against FRß(+) AML in vitro and in vivo compared with a low-affinity FRß CAR (54.3 nm KD). Using the HA-FRß immunoglobulin G, FRß expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34(+) hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRß CAR T cells lysed mature CD14(+) monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRß CAR T cells retained effective antitumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRß CAR T cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.
Assuntos
Receptor 2 de Folato/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linhagem Celular , Linhagem da Célula , Células-Tronco Hematopoéticas , Humanos , Leucemia Mieloide Aguda , Camundongos , Camundongos Transgênicos , Monócitos , Células Mieloides , Anticorpos de Cadeia Única , Linfócitos T/imunologiaAssuntos
Benzoatos/uso terapêutico , Hidrazinas/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Pirazóis/uso terapêutico , Receptores de Trombopoetina/agonistas , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/patologia , Receptores de Trombopoetina/análise , Receptores de Trombopoetina/fisiologiaRESUMO
Abnormal gene expression is a hallmark of many diseases. Gene-specific downregulation of aberrant genes could be useful therapeutically and potentially less toxic than conventional therapies due its specificity. Over the years, many strategies have been proposed for silencing gene expression in a gene-specific manner. Three major approaches are antisense oligonucleotides (AS-ONs), ribozymes/DNAzymes, and RNA interference (RNAi). In this brief review, we will discuss the successes and shortcomings of these three gene-silencing methods, and the approaches being taken to improve the effectiveness of antisense molecules. We will also provide an overview of some of the clinical applications of antisense therapy.
Assuntos
Ácidos Nucleicos/uso terapêutico , RNA Mensageiro/uso terapêutico , Animais , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/patologia , Inativação Gênica , Humanos , Imunoterapia , Inflamação/tratamento farmacológico , Inflamação/patologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , RNA Antissenso/uso terapêuticoRESUMO
To be effective in vivo, antisense oligonucleotides (AS ON) should be nuclease resistant, form stable ON/RNA duplexes and support ribonuclease H mediated heteroduplex cleavage, all with negligible non-specific effects on cell function. We report herein that AS ONs containing a 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F-ANA) sugar modification not only meet these criteria, but have the added advantage of maintaining high intracellular concentrations for prolonged periods of time which appears to promote longer term gene silencing. To demonstrate this, we targeted the c-MYB protooncogene's mRNA in human leukemia cells with fully phosphorothioated 2'F-ANA-DNA chimeras (PS-2'FANA-DNA) and compared their gene silencing efficiency with AS ON containing unmodified nucleosides (PS-DNA). When delivered by nucleofection, chemically modified ON of both types effected a >90% knockdown of c-MYB mRNA and protein expression, but the PS-2'F-ANA-DNA were able to accomplish this at 20% of the dose of the PS-DNA, and in contrast to the PS-AS DNA, their silencing effect was still present after 4 days after a single administration. Therefore, our data demonstrate that PS-2'F-ANA-DNA chimeras are efficient gene silencing molecules, and suggest that they could have significant therapeutic potential.
Assuntos
Arabinonucleotídeos/química , Inativação Gênica , Oligodesoxirribonucleotídeos Antissenso/química , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Humanos , Células K562 , Cinética , Oligodesoxirribonucleotídeos Antissenso/metabolismo , Proteínas Proto-Oncogênicas c-myb/biossíntese , Proteínas Proto-Oncogênicas c-myb/genética , Tionucleotídeos/química , Tionucleotídeos/metabolismo , Tionucleotídeos/farmacologiaRESUMO
Incorporation of nucleosides with novel base-constraining oxetane (OXE) modifications [oxetane, 1-(1',3'-O-anhydro-beta-d-psicofuranosyl nucleosides)] into antisense (AS) oligodeoxyribonucleotides (ODNs) should greatly improve the gene silencing efficiency of these molecules. This is because OXE modified bases provide nuclease protection to the natural backbone ODNs, can impart T(m) values similar to those predicted for RNA-RNA hybrids, and not only permit but also accelerate RNase H mediated catalytic activity. We tested this assumption in living cells by directly comparing the ability of OXE and phosphorothioate (PS) ODNs to target c-myb gene expression. The ODNs were targeted to two different sites within the c-myb mRNA. One site was chosen arbitrarily. The other was a 'rational' choice based on predicted hybridization accessibility after physical mapping with self-quenching reporter molecules (SQRM). The Myb mRNA and protein levels were equally diminished by OXE and PS ODNs, but the latter were delivered to cells with approximately six times greater efficiency, suggesting that OXE modified ODNs were more potent on a molar basis. The rationally targeted molecules demonstrated greater silencing efficiency than those directed to an arbitrarily chosen mRNA sequence. We conclude that rationally targeted, OXE modified ODNs, can function efficiently as gene silencing agents, and hypothesize that they will prove useful for therapeutic purposes.