A novel non-viral delivery method that enables efficient engineering of primary human T cells for ex vivo cell therapy applications.

BACKGROUND AIMS: Next-generation immune cell therapy products will require complex modifications using engineering technologies that can maintain high levels of cell functionality. Non-viral engineering methods have the potential to address limitations associated with viral vectors. However, while electroporation is the most widely used non-viral modality, concerns about its effects on cell functionality have led to the exploration of alternative approaches. Here the authors have examined the suitability of the Solupore non-viral delivery system for engineering primary human T cells for cell therapy applications. METHODS: The Solupore system was used to deliver messenger RNA (mRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) guide RNA ribonucleoprotein (RNP) cargos to T cells, and efficiency was measured by flow cytometry. Cell perturbation was assessed by immune gene expression profiling, including an electroporation comparator. In vitro and in vivo cytotoxicity of chimeric antigen receptor (CAR) T cells generated using the Solupore system was evaluated using a real-time cellular impedance assay and a Raji-luciferase mouse tumor model, respectively. RESULTS: Efficient transfection was demonstrated through delivery of mRNA and CRISPR CAS9 RNP cargos individually, simultaneously and sequentially using the Solupore system while consistently maintaining high levels of cell viability. Gene expression profiling revealed minimal alteration in immune gene expression, demonstrating the low level of perturbation experienced by the cells during this transfection process. By contrast, electroporation resulted in substantial changes in immune gene expression in T cells. CAR T cells generated using the Solupore system exhibited efficient cytotoxicity against target cancer cells in vitro and in vivo. CONCLUSIONS: The Solupore system is a non-viral means of simply, rapidly and efficiently delivering cargos to primary human immune cells with retention of high cell viability and functionality.

Macular pigment and percentage of body fat.

PURPOSE: To investigate the relationship between percentage of body fat and macular pigment (MP) optical density. METHODS: One hundred healthy subjects of ages between 22 and 60 years volunteered to participate in this study. MP optical density was measured psychophysically, serum lutein and zeaxanthin were quantified by HPLC, and dietary intake of lutein and zeaxanthin was assessed using a validated food frequency questionnaire. Body fat was measured by dual energy x-ray absorptiometry (DEXA); body mass index (BMI) was also calculated for each subject. Clinical and personal details were recorded, with particular attention directed toward putative risk factors for AMD. RESULTS: There was a significant inverse relationship between the percentage of body fat and MP optical density in males (r=-0.392, P <0.01), and after correcting for age and dietary lutein and zeaxanthin, this inverse relationship remained significant (r=-0.290, P <0.05). The relationship between MP optical density and percentage of body fat in females was inverse, but not significant (r=-0.197, P=0.149). A significant and inverse relationship between serum zeaxanthin and percentage of body fat was observed for females only (r=-0.354, P <0.01). Dietary intake of fat was inversely related to serum lutein and zeaxanthin, and significantly so for lutein (r=-0.256, P <0.05). However, dietary fat was unrelated to MP optical density (r=0.041, P=0.688). CONCLUSIONS: A relative lack of MP is associated with adiposity in men, and may underlie the association between body fat and risk for AMD progression in males. Further, the processes governing accumulation and/or stabilization of lutein and zeaxanthin in fat tissue appear to differ for males and females.

Macular pigment optical density and its relationship with serum and dietary levels of lutein and zeaxanthin.

Observational evidence is accumulating that the onset of age-related maculopathy, the leading cause of legal blindness in the Western World, could be delayed, or even averted, with antioxidant supplements. Lutein (L) and zeaxanthin (Z) are two hydroxy-carotenoids with antioxidant activity which accumulate at the macula, where they are collectively known as macular pigment (MP). It has been shown that MP is entirely of dietary origin, and that L and Z levels in serum, diet, and retina correlate. However, the nature of the relationships between L and Z in foodstuffs, blood, and macula is confounded by many variables including processes which influence digestion, absorption, and transport of the compounds in question, and accumulation and stabilization of the carotenoids in the tissues. If macular pigment is protective for age-related maculopathy, a clear understanding of the mechanisms whereby L and Z arrive at the target tissue (retina) from their source (foodstuff) is essential. In this paper, we review the literature germane to this growing area of interest.