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

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.

%B Elife %V 11 %8 2022 02 11 %G eng %R 10.7554/eLife.65421 %0 Journal Article %J J Med Virol %D 2022 %T Omicron infection increases IgG binding to spike protein of predecessor variants. %A Mahalingam, Gokulnath %A Periyasami, Yogapriya %A Arjunan, Porkizhi %A Subaschandrabose, Rajesh Kumar %A Mathivanan, Tamil Venthan %A Mathew, Roshlin Susan %A Ramya Devi, Kt %A Premkumar, Prasanna Samuel %A Muliyil, Jayaprakash %A Srivastava, Alok %A Moorthy, Mahesh %A Marepally, Srujan %X

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.

%B J Med Virol %8 2022 Dec 22 %G eng %R 10.1002/jmv.28419 %0 Journal Article %J ACS Appl Mater Interfaces %D 2022 %T Skin-Permeable Nano-Lithocholic Lipidoid Efficiently Alleviates Psoriasis-like Chronic Skin Inflammations. %A Rachamalla, Hari Krishnareddy %A Voshavar, Chandrashekhar %A Arjunan, Porkizhi %A Mahalingam, Gokulnath %A Chowath, Rashmi Praksash %A Banerjee, Rajkumar %A Vemula, Praveen Kumar %A Marepally, Srujan %X

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.

%B ACS Appl Mater Interfaces %8 2022 Mar 29 %G eng %R 10.1021/acsami.1c19180