RESUMEN
Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Staphylococcus/enzimología , Animales , Proteína 9 Asociada a CRISPR/aislamiento & purificación , Línea Celular Tumoral , Dependovirus , Modelos Animales de Enfermedad , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Ratones , Parvovirinae/genética , Ingeniería de Proteínas , Ribonucleasas , Staphylococcus/genética , Especificidad por Sustrato , Síndromes de Usher/genética , Síndromes de Usher/terapia , ARN Guía de Sistemas CRISPR-CasRESUMEN
The present study aims to evaluate the efficacy of octa-arginine (R8)-modified pegylated liposomal doxorubicin (R8-PLD) for the treatment of non-small cell lung cancer, for which the primary treatment modality currently consists of surgery and radiotherapy. Cell-penetrating peptide R8 modification of Doxorubicin-(Dox)-loaded liposomes was performed by post-insertion of an R8-conjugated amphiphilic PEG-PE copolymer (R8-PEG-DOPE) into the liposomal lipid bilayer. In vitro analysis with the non-small cell lung cancer cell line, A549 confirmed the efficient cellular accumulation of Dox, delivered by R8-PLD compared to PLD. It led to the early initiation of apoptosis and a 9-fold higher level of the apoptotic regulator, caspase 3/7 (9.24±0.34) compared to PLD (1.07±0.19) at Dox concentration of 100 µg/mL. The treatment of A549 monolayers with R8-PLD increased the level of cell death marker lactate dehydrogenase (LDH) secretion (1.2±0.1 for PLD and 2.3±0.1 for R8-PLD at Dox concentration of 100 µg/mL) confirming higher cytotoxicity of R8-PLD than PLD, which was ineffective under the same treatment regimen (cell viability 90±6% in PLD vs. 45±2% in R8-PLD after 24h). R8-PLD had significantly higher penetration into the hypoxic A549 tumor spheroids compared to PLD. R8-PLD induced greater level of apoptosis to A549 tumor xenograft and dramatic inhibition of tumor volume and tumor weight reduction. The R8-PLD treated tumor lysate had a elevated caspase 3/7 expression than with R8-PLD treatment. This suggested system improved the delivery efficiency of Dox in selected model of cancer which supports the potential usefulness of R8-PLD in cancer treatment, lung cancer in particular.
