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1.
Proc Natl Acad Sci U S A ; 118(45)2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34732575

RESUMO

Triplex gene editing relies on binding a stable peptide nucleic acid (PNA) sequence to a chromosomal target, which alters the helical structure of DNA to stimulate site-specific recombination with a single-strand DNA (ssDNA) donor template and elicits gene correction. Here, we assessed whether the codelivery of PNA and donor template encapsulated in Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles can correct sickle cell disease and x-linked severe combined immunodeficiency. However, through this process we have identified a false-positive PCR artifact due to the intrinsic capability of PNAs to aggregate with ssDNA donor templates. Here, we show that the combination of PNA and donor templates but not either agent alone results in different degrees of aggregation that result in varying but highly reproducible levels of false-positive signal. We have identified this phenomenon in vitro and confirmed that the PNA sequences producing the highest supposed correction in vitro are not active in vivo in both disease models, which highlights the importance of interrogating and eliminating carryover of ssDNA donor templates in assessing various gene editing technologies such as PNA-mediated gene editing.


Assuntos
Edição de Genes/métodos , Anemia Falciforme/genética , Animais , Reações Falso-Positivas , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos SCID , Técnicas de Sonda Molecular , Ácidos Nucleicos Peptídicos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico
2.
Infect Immun ; 86(9)2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29967089

RESUMO

T cell exhaustion is a state of hyporesponsiveness that develops during many chronic infections and cancer. Neutralization of inhibitory receptors, or "checkpoint blockade," can reverse T cell exhaustion and lead to beneficial prognoses in experimental and clinical settings. Whether checkpoint blockade can resolve lethal acute infections is less understood but may be beneficial in vaccination protocols that fail to elicit sterilizing immunity. Since a fully protective vaccine for any human parasite has yet to be developed, we explored the efficacy of checkpoint inhibitors in a mouse model of Toxoplasma gondii reinfection. Mice chronically infected with an avirulent type III strain survive reinfection with the type I RH strain but not the MAS, GUY-DOS, and GT1 parasite strains. We report here that mouse susceptibility to secondary infection correlates with the initial parasite burden and that protection against the RH strain is dependent on CD8 but not CD4 T cells in this model. When given a lethal secondary infection, CD8 and CD4 T cells upregulate several coinhibitory receptors, including PD-1, TIM-3, 4-1bb, and CTLA-4. Moreover, the gamma interferon (IFN-γ) response of CD8 but not CD4 T cells is significantly reduced during secondary infection with virulent strains, suggesting that checkpoint blockade may reduce disease severity. However, single and combination therapies targeting TIM-3, CTLA-4, and/or PD-L1 failed to reverse susceptibility to secondary infection. These results suggest that additional host responses, which are refractory to checkpoint blockade, are likely required for immunity to this pathogen.


Assuntos
Antígeno B7-H1/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Toxoplasma/patogenicidade , Toxoplasmose Animal/imunologia , Animais , Modelos Animais de Doenças , Feminino , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
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