TY - JOUR T1 - Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin. JF - Elife Y1 - 2022 A1 - Ravi, Nithin Sam A1 - Wienert, Beeke A1 - Wyman, Stacia K A1 - Bell, Henry William A1 - George, Anila A1 - Mahalingam, Gokulnath A1 - Vu, Jonathan T A1 - Prasad, Kirti A1 - Bandlamudi, Bhanu Prasad A1 - Devaraju, Nivedhitha A1 - Rajendiran, Vignesh A1 - Syedbasha, Nazar A1 - Pai, Aswin Anand A1 - Nakamura, Yukio A1 - Kurita, Ryo A1 - Narayanasamy, Muthuraman A1 - Balasubramanian, Poonkuzhali A1 - Thangavel, Saravanabhavan A1 - Marepally, Srujan A1 - Velayudhan, Shaji R A1 - Srivastava, Alok A1 - DeWitt, Mark A A1 - Crossley, Merlin A1 - Corn, Jacob E A1 - Mohankumar, Kumarasamypet M KW - Adenine KW - Anemia, Sickle Cell KW - beta-Globins KW - beta-Thalassemia KW - Cell Line KW - Clustered Regularly Interspaced Short Palindromic Repeats KW - CRISPR-Cas Systems KW - Cytosine KW - Fetal Hemoglobin KW - gamma-Globins KW - Gene Editing KW - Hematopoietic Stem Cells KW - Humans KW - Point Mutation KW - Promoter Regions, Genetic AB -

Naturally occurring point mutations in the promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

VL - 11 ER - TY - JOUR T1 - Omicron infection increases IgG binding to spike protein of predecessor variants. JF - J Med Virol Y1 - 2022 A1 - Mahalingam, Gokulnath A1 - Periyasami, Yogapriya A1 - Arjunan, Porkizhi A1 - Subaschandrabose, Rajesh Kumar A1 - Mathivanan, Tamil Venthan A1 - Mathew, Roshlin Susan A1 - Ramya Devi, Kt A1 - Premkumar, Prasanna Samuel A1 - Muliyil, Jayaprakash A1 - Srivastava, Alok A1 - Moorthy, Mahesh A1 - Marepally, Srujan AB -

BACKGROUND: SARS-CoV-2 transmission in India in 2020-2022 was driven predominantly by Wild (Wuhan-Hu-1and D614G), Delta, and Omicron variants. The aim of this study was to examine the effect of infections on the humoral immune response and cross-reactivity to spike proteins of Wuhan-Hu-1, Delta, C.1.2., and Omicron.

OBJECTIVES: Residual archival sera (N=81) received between January 2020 and March 2022 were included. Infection status was inferred by a positive SARS-CoV-2 RT-PCR and/or serology (anti-N and anti-S antibodies) and sequencing of contemporaneous samples (N=18) to infer lineage. We estimated the levels and cross-reactivity of infection-induced sera including Wild, Delta, Omicron as well as vaccine breakthrough infections (Delta and Omicron).

RESULTS: We found ~2-fold increase in spike-specific IgG antibody binding in post-Omicron infection compared to the pre-Omicron period, whilst the change in pre- and post-Delta infections were similar. Further investigation of Omicron-specific humoral responses revealed primary Omicron infection as an inducer of cross-reactive antibodies against predecessor variants, in spite of weaker degree of humoral response compared to Wuhan-Hu-1 and Delta infection. Intriguingly, Omicron vaccine-breakthrough infections when compared with primary infections, exhibited increased humoral responses against RBD (7.7-fold) and Trimeric S (Trimeric form of spike protein) (34.6-fold) in addition to increased binding of IgGs towards previously circulating variants (4.2 - 6.5-fold). Despite Delta breakthrough infections showing a higher level of humoral response against RBD (2.9-fold) and Trimeric S (5.7-fold) compared to primary Delta sera, a demonstrably reduced binding (36-49%) was observed to Omicron spike protein.

CONCLUSIONS: Omicron vaccine breakthrough infection results in increased intensity of humoral response and wider breadth of IgG binding to spike proteins of antigenically-distinct, predecessor variants. This article is protected by copyright. All rights reserved.

ER - TY - JOUR T1 - Skin-Permeable Nano-Lithocholic Lipidoid Efficiently Alleviates Psoriasis-like Chronic Skin Inflammations. JF - ACS Appl Mater Interfaces Y1 - 2022 A1 - Rachamalla, Hari Krishnareddy A1 - Voshavar, Chandrashekhar A1 - Arjunan, Porkizhi A1 - Mahalingam, Gokulnath A1 - Chowath, Rashmi Praksash A1 - Banerjee, Rajkumar A1 - Vemula, Praveen Kumar A1 - Marepally, Srujan AB -

Long-term application of topical therapeutics for psoriasis has a plethora of side effects. Additionally, skin-permeating agents used in their formulations for deeper dermal delivery damage the skin. To address these limitations, we developed novel lithocholic acid analogues that could form lipid nanoparticles (nano-LCs) spontaneously in the aqueous milieu, permeate through the skin, penetrate the deeper dermal layers, and exert anti-inflammatory effects against psoriasis-like chronic skin inflammations. Prior findings demonstrated that lithocholic acid acts as a vitamin D receptor agonist without affecting the Ca metabolism and also as an antagonist for ephrin type-A receptor 2 (EphA2). Taking cues from the previous findings, lithocholic acid derivatives with twin alkyl chains (LC6, LC8, LC10, and LC-12) were synthesized, nanoparticles (nano-LCs) were prepared, and they were evaluated for their skin permeability and anti-inflammatory properties. Among these nano-LCs, nano-LC10 demonstrated superior anti-inflammatory properties and inhibition of keratinocyte proliferation in various cell-based evaluations. Furthermore, the therapeutic efficiency of nano-LC10 was evaluated in an imiquimod-induced psoriasis-like mouse model and demonstrated comparable efficiency with the standard topical formulation, Sorvate, in reducing skin inflammations. Nano-LC10 also reduced systemic inflammation, organ toxicity, and also proinflammatory serum cytokine levels. Overall, nano-lithocholic lipidoid (nano-LC10) can be a potential novel class of therapeutics for topical application in treating psoriasis.

ER -