bims-limsir 2022-07-10 papers

on Lipophilic modified siRNAs

Issue of 2022–07–10
nine papers selected by
Ivan V. Chernikov, Institute of Сhemical Biology and Fundamental Medicine of the SB RAS

PK-modifying anchors significantly alter clearance kinetics, tissue distribution, and efficacy of therapeutics siRNAs.
Antisense Oligonucleotides and Small Interfering RNA for the Treatment of Dyslipidemias.
Chemical Modifications of CRISPR RNAs to Improve Gene-Editing Activity and Specificity.
Improved anti-solid tumor response by humanized anti-podoplanin chimeric antigen receptor transduced human cytotoxic T cells in an animal model.
Barriers and Opportunities for CAR T-Cell Targeting of Solid Tumors.
The molecular mechanism of microRNA duplex selectivity of Arabidopsis ARGONAUTE10.
Swarms of chemically modified antiviral siRNA targeting herpes simplex virus infection in human corneal epithelial cells.
Identification of nucleobase chemical modifications that reduce the hepatotoxicity of gapmer antisense oligonucleotides.
Clinical trials for chimeric antigen receptor T-cell therapy: lessons learned and future directions.

Mol Ther Nucleic Acids. 2022 Sep 13. 29 116-132

PK-modifying anchors significantly alter clearance kinetics, tissue distribution, and efficacy of therapeutics siRNAs.

Bruno M D C Godinho, Emily G Knox, Samuel Hildebrand, James W Gilbert, Dimas Echeverria, Zachary Kennedy, Reka A Haraszti, Chantal M Ferguson, Andrew H Coles, Annabelle Biscans, Jillian Caiazzi, Julia F Alterman, Matthew R Hassler, Anastasia Khvorova.

  Effective systemic delivery of small interfering RNAs (siRNAs) to tissues other than liver remains a challenge. siRNAs are small (∼15 kDa) and therefore rapidly cleared by the kidneys, resulting in limited blood residence times and tissue exposure. Current strategies to improve the unfavorable pharmacokinetic (PK) properties of siRNAs rely on enhancing binding to serum proteins through extensive phosphorothioate modifications or by conjugation of targeting ligands. Here, we describe an alternative strategy for enhancing blood and tissue PK based on dynamic modulation of the overall size of the siRNA. We engineered a high-affinity universal oligonucleotide anchor conjugated to a high-molecular-weight moiety, which binds to the 3' end of the guide strand of an asymmetric siRNA. Data showed a strong correlation between the size of the PK-modifying anchor and clearance kinetics. Large 40-kDa PK-modifying anchors reduced renal clearance by ∼23-fold and improved tissue exposure area under the curve (AUC) by ∼26-fold, resulting in increased extrahepatic tissue retention (∼3- to 5-fold). Furthermore, PK-modifying oligonucleotide anchors allowed for straightforward and versatile modulation of blood residence times and biodistribution of a panel of chemically distinct ligands. The effects were more pronounced for conjugates with low lipophilicity (e.g., N-Acetylgalactosamine [GalNAc]), where significant improvement in uptake by hepatocytes and dose-dependent silencing in the liver was observed.

Keywords:  GalNAc; Gene silencing; PEGylation; RNA interference; extrahepatic; oligonucleotide; siRNA conjugates

DOI:  https://doi.org/10.1016/j.omtn.2022.06.005
J Clin Med. 2022 Jul 04. pii: 3884. [Epub ahead of print]11(13):

Antisense Oligonucleotides and Small Interfering RNA for the Treatment of Dyslipidemias.

Clarice Gareri, Alberto Polimeni, Salvatore Giordano, Laura Tammè, Antonio Curcio, Ciro Indolfi.

  The burden of atherosclerotic disease worldwide necessitates implementing the treatment of its risk factors. Among them, hypercholesterolemia has a central role. In addition to conventional small organic compounds and the recently introduced monoclonal antibodies, new technologies are arising such as the antisense oligonucleotides and small interfering RNAs (siRNAs) that operate upstream, blocking the mRNA translation of the proteins specifically involved in lipid metabolism. In this review, we briefly explain the mechanisms of action of these molecules and discuss the difficulties related to their in vivo use as therapeutical agents. We go over the oligonucleotides tested in clinical trials that could potentially revolutionize the care of patients by acting on proteins involved in the lipoprotein metabolism and regulation, namely: angiopoietin-like protein 3 (ANGPTL3); lipoprotein a (Lp(a)); apolipoprotein B (Apo B); apolipoprotein C III (Apo C-III); and proprotein convertase subtilisin-kexin type 9 (PCSK9). Finally, the differences between ASOs and siRNAs, their future possible clinical applications, and the role of Inclisiran, a siRNA direct against PCSK9 to reduce LDL-C, were reviewed in detail.

Keywords:  ASO; hypercholesterolemia; miRNA; siRNAF

DOI:  https://doi.org/10.3390/jcm11133884
J Am Chem Soc. 2022 Jul 07.

Chemical Modifications of CRISPR RNAs to Improve Gene-Editing Activity and Specificity.

Eriks Rozners.

CRISPR (clustered, regularly interspaced, short palindromic repeats) has become a cutting-edge research method and holds great potential to revolutionize biotechnology and medicine. However, like other nucleic acid technologies, CRISPR will greatly benefit from chemical innovation to improve activity and specificity for critical in vivo applications. Chemists have started optimizing various components of the CRISPR system; the present Perspective focuses on chemical modifications of CRISPR RNAs (crRNAs). As with other nucleic acid-based technologies, early efforts focused on well-established sugar and backbone modifications (2'-deoxy, 2'-F, 2'-OMe, and phosphorothioates). Some more significant alterations of crRNAs have been done using bicyclic (locked) riboses and phosphate backbone replacements (phosphonoacetates and amides); however, the range of chemical innovation applied to crRNAs remains limited to modifications that have been successful in RNA interference and antisense technologies. The encouraging results given by these tried-and-true modifications suggest that, going forward, chemists should take a bolder approach─research must aim to investigate what chemistry will have the most impact on maturing CRISPR as therapeutic and other in vivo technologies. With an eye to the future, this Perspective argues that the complexity of CRISPR presents rich unprecedented opportunities for chemists to synergize advances in synthetic methodology and structural biochemistry to rationally optimize crRNA-protein interactions.

DOI: https://doi.org/10.1021/jacs.2c02633
Genes Cells. 2022 Jul 05.

Improved anti-solid tumor response by humanized anti-podoplanin chimeric antigen receptor transduced human cytotoxic T cells in an animal model.

Akihiro Ishikawa, Masazumi Waseda, Tomoko Ishii, Mika K Kaneko, Yukinari Kato, Shin Kaneko.

  Recently, research has been conducted with chimeric antigen receptor (CAR)-T cells to improve efficacy against solid tumors. Humanized CAR improved the long-term survival of CAR-T cells in patients' peripheral blood, resulting in increased therapeutic efficacy. Therefore, the humanization of the CAR-gene sequence is considered an effective method. Podoplanin (PDPN) is a glycosylated transmembrane protein that is highly expressed in solid tumors and is associated with poor prognosis in patients with cancer. Therefore, PDPN is considered a biomarker and good target for cancer treatment with CAR-T cells. Previously, an anti-PDPN CAR was generated from a conventional non-humanized antibody-NZ-1, the only anti-PDPN antibody for which a CAR was produced. In this study, we investigated other anti-PDPN CARs from the antibody NZ-27, or humanized NZ-1, to enhance the therapeutic potential of CAR-T cells. The CAR signal intensity was enhanced by the efficient expression of CAR proteins on the T-cell surface of NZ-27 CAR-T cells, which show tumor-specific cytotoxicity, proinflammatory cytokine production, and anti-tumor activity against PDPN-expressing tumor xenografts in mice that were significantly better than those in non-humanized NZ-1 CAR-T cells. This article is protected by copyright. All rights reserved.

Keywords:  CAR-T cells; chimeric antigen receptor; humanized antibody; podoplanin; solid tumor

DOI:  https://doi.org/10.1111/gtc.12972
Immunol Invest. 2022 Jul 07. 1-11

Barriers and Opportunities for CAR T-Cell Targeting of Solid Tumors.

Jose R Conejo-Garcia, Jose A Guevara-Patino.

  CAR T-cell therapy has transformed the treatment of hematological malignancies of the B cell lineage. However, the quest to fulfil the same promise for solid tumors is still in its infancy. This review summarizes some of the challenges that the field is trying to overcome for effective treatment of human carcinomas, including tumor heterogeneity, the paucity of truly tumor-specific targets, immunosuppression and metabolic restrictions at solid tumor beds, and defective T-cell trafficking. All these barriers are being currently investigated and, in some cases, targeted, by multiple independent groups. With clinical interventions against multiple human malignancies and different platforms under accelerated clinical development, the next few years will see an array of cellular therapies, including CAR T-cells, progressively becoming routine interventions to eliminate currently incurable diseases, as it happened with some hematological malignancies.

Keywords:  Carcinoma; T cell; chimeric antigen receptor; immunosuppression; immunotherapy

DOI:  https://doi.org/10.1080/08820139.2022.2096463
Nucleic Acids Res. 2022 Jul 08. pii: gkac571. [Epub ahead of print]

The molecular mechanism of microRNA duplex selectivity of Arabidopsis ARGONAUTE10.

Yao Xiao, Ian J MacRae.

Small RNAs (sRNAs), including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are essential gene regulators for plant and animal development. The loading of sRNA duplexes into the proper ARGONAUTE (AGO) protein is a key step to forming a functional silencing complex. In Arabidopsis thaliana, the specific loading of miR166/165 into AGO10 (AtAGO10) is critical for the maintenance of the shoot apical meristem, the source of all shoot organs, but the mechanism by which AtAGO10 distinguishes miR166/165 from other cellular miRNAs is not known. Here, we show purified AtAGO10 alone lacks loading selectivity towards miR166/165 duplexes. However, phosphate and HSP chaperone systems reshape the selectivity of AtAGO10 to its physiological substrates. A loop in the AtAGO10 central cleft is essential for recognizing specific mismatches opposite the guide strand 3' region in miR166/165 duplexes. Replacing this loop with the equivalent loop from Homo sapiens AGO2 (HsAGO2) changes AtAGO10 miRNA loading behavior such that 3' region mismatches are ignored and mismatches opposite the guide 5' end instead drive loading, as in HsAGO2. Thus, this study uncovers the molecular mechanism underlying the miR166/165 selectivity of AtAGO10, essential for plant development, and provides new insights into how miRNA duplex structures are recognized for sRNA sorting.

DOI: https://doi.org/10.1093/nar/gkac571
PLoS Pathog. 2022 Jul 06. 18(7): e1010688

Swarms of chemically modified antiviral siRNA targeting herpes simplex virus infection in human corneal epithelial cells.

Kiira Kalke, Liisa M Lund, Marie C Nyman, Alesia A Levanova, Arto Urtti, Minna M Poranen, Veijo Hukkanen, Henrik Paavilainen.

Herpes simplex virus type 1 (HSV-1) is a common virus of mankind and HSV-1 infections are a significant cause of blindness. The current antiviral treatment of herpes infection relies on acyclovir and related compounds. However, acyclovir resistance emerges especially in the long term prophylactic treatment that is required for prevention of recurrent herpes keratitis. Earlier we have established antiviral siRNA swarms, targeting sequences of essential genes of HSV, as effective means of silencing the replication of HSV in vitro or in vivo. In this study, we show the antiviral efficacy of 2´-fluoro modified antiviral siRNA swarms against HSV-1 in human corneal epithelial cells (HCE). We studied HCE for innate immunity responses to HSV-1, to immunostimulatory cytotoxic double stranded RNA, and to the antiviral siRNA swarms, with or without a viral challenge. The panel of studied innate responses included interferon beta, lambda 1, interferon stimulated gene 54, human myxovirus resistance protein A, human myxovirus resistance protein B, toll-like receptor 3 and interferon kappa. Our results demonstrated that HCE cells are a suitable model to study antiviral RNAi efficacy and safety in vitro. In HCE cells, the antiviral siRNA swarms targeting the HSV UL29 gene and harboring 2´-fluoro modifications, were well tolerated, induced only modest innate immunity responses, and were highly antiviral with more than 99% inhibition of viral release. The antiviral effect of the 2'-fluoro modified swarm was more apparent than that of the unmodified antiviral siRNA swarm. Our results encourage further research in vitro and in vivo on antiviral siRNA swarm therapy of corneal HSV infection, especially with modified siRNA swarms.

DOI: https://doi.org/10.1371/journal.ppat.1010688
Nucleic Acids Res. 2022 Jul 08. pii: gkac562. [Epub ahead of print]

Identification of nucleobase chemical modifications that reduce the hepatotoxicity of gapmer antisense oligonucleotides.

Tokuyuki Yoshida, Kunihiko Morihiro, Yuki Naito, Atsushi Mikami, Yuuya Kasahara, Takao Inoue, Satoshi Obika.

Currently, gapmer antisense oligonucleotide (ASO) therapeutics are under clinical development for the treatment of various diseases, including previously intractable human disorders; however, they have the potential to induce hepatotoxicity. Although several groups have reported the reduced hepatotoxicity of gapmer ASOs following chemical modifications of sugar residues or internucleotide linkages, only few studies have described nucleobase modifications to reduce hepatotoxicity. In this study, we introduced single or multiple combinations of 17 nucleobase derivatives, including four novel derivatives, into hepatotoxic locked nucleic acid gapmer ASOs and examined their effects on hepatotoxicity. The results demonstrated successful identification of chemical modifications that strongly reduced the hepatotoxicity of gapmer ASOs. This approach expands the ability to design gapmer ASOs with optimal therapeutic profiles.

DOI: https://doi.org/10.1093/nar/gkac562
Curr Opin Hematol. 2022 Jul 01. 29(4): 225-232

Clinical trials for chimeric antigen receptor T-cell therapy: lessons learned and future directions.

Brett A Schroeder, Jennifer Jess, Hari Sankaran, Nirali N Shah.

PURPOSE OF REVIEW: The purpose of this review is to summarize the status and utilization of chimeric antigen receptor T-cell (CAR-T) therapy based on the most recent clinical trials in patients with leukemia and lymphoma. Additionally, this review will highlight limitations in current strategies, discuss efforts in toxicity mitigation, and outline future directions for investigation.
RECENT FINDINGS: CD19 targeted CAR-T-cell therapy (CD19-CAR) is highly effective in patients with relapsed/refractory (r/r) B-cell hematologic malignancies. However, multiple challenges have arisen, particularly life-threatening adverse events, such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. Despite these challenges, recent CD19-CAR trials, including two randomized studies, have demonstrated both impressive initial results along with durable responses. Combined with results emerging from 'real-world' experience, the efficacy of CAR-T-cells is high, propelling CAR-T-cells studies targeting alternate B-cell antigens [e.g. CD20, CD22 and CD269 (BCMA)] and other targets for hematologic malignancies, along with solid and CNS tumors.
SUMMARY: Given the benefit for CD19-CAR, determining the appropriate place in utilization for both an individual patient's treatment course and more broadly in the generalized treatment paradigm is critically needed. We discuss the most recent trials exploring this topic and future directions in the field.

DOI: https://doi.org/10.1097/MOH.0000000000000723