Perspectives of Genetic Management Strategy for Inherited Cardiovascular Diseases in China

Inherited cardiovascular diseases (CVDs) threaten human health and pose an enormous economic burden worldwide. Genetic alteration is a major risk factor for many CVDs. These disorders are usually controlled by a pair of alleles, affecting offspring according to the Mendelian principle, regardless of isolated primary damage or secondary injury from other syndromes or deficiency. To date, there are hundreds of inherited CVDs. With advances in next-generation sequencing (NGS) technologies, rapid and accurate molecular diagnosis of patients with inherited CVDs is clinically practical. Besides, great improvements have been made in recent years, and targeted therapy and assist devices have been used in clinical practice. Yet there is still no totally efficient strategy for dealing with inherited CVDs. Accordingly, there is an urgent need to develop better therapeutic strategies for treating inherited CVDs.[1] Heritable genetic disorders occur among an estimated 1 in 50 living newborns. The question of how to restore the impaired gene has been studied for more than three decades. Currently, the major obstacle in developing gene therapy for inherited diseases is determining how to restore gene function most efficiently and safely. Novel approaches for gene therapy. (1) Gene addition/replacement. In the late 1980s, intracoronary artery gene transfer paved the way for gene therapy for CVDs. Technology in this field has exploded in the past 20 years. In the beginning, strategies were developed to deliver new copies of functional genes. This created the idea of gene replacement. The main idea behind gene replacement is to replace the impaired gene with new nucleotide sequences in either RNA or DNA form using viral or non-viral strategies. As delivery vectors improved, gene addition with non-homologous recombination became more acceptable for generating new copies of functional genes in vivo. Wang et al[2] and Thompson et al[3] demonstrated the re-expression of the Tafazzin gene using an adeno-associated virus (AAV) vector in a mouse model of Barth syndrome to improve contractile function in both cardiac and skeletal myocytes. However, as the size of the vector is limited, it is impossible to deliver very large genes. Moreover, it is rare that additional genes are able to be regulated by their original promote. (2) Gene inhibition/depletion. Gene inhibition is involved in several diseases. Antisense oligonucleotide (ASO), small interfering RNA, and selected micro-RNA have been used in translational research and even in clinical trials, to realize gene inhibition in vivo. Inclisiran (ALN-PCSsc), approved for the use in a clinical trial, is a long-acting RNA interference (RNAi) therapeutic agent that inhibits the synthesis of proprotein convertase subtilisin–kexin type 9 (Pcsk9), a target for lowering low-density lipoprotein (LDL) cholesterol to prevent coronary heart disease.[4] In a phase 3 trial, inclisiran significantly reduced LDL cholesterol levels by approximately 50% for at least 6 months.[5] Other RNAi products, for instance have already been approved for clinical treatment. Unfortunately, no product of RNAi is available to treat inherited CVDs. (3) Gene editing. Gene editing is an alternative method of gene therapy. Zinc finger nucleases and transcription activator-like effector nucleases (TALENs) made it possible to perform gene editing in vivo for clinical treatment 10 years ago. However, developing these tools is time and resource intensive, and the resulting candidate editing agents can vary substantially in activity and specificity for the targeted site. (4) Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein 9 (Cas9). Gene editing achieved through CRISPR/Cas9-based NHEJ has been used in clinical trials. NTLA-2001 is one of the first gene editing therapeutic agents clinically approved to treat transthyretin amyloidosis based on CRISPR/Cas9-mediated NHEJ by markedly reducing a disease-causing protein, misfolded transthyretin (TTR). Data from the phase I clinical trial showed that up to 97% serum TTR reduction was maintained for 12 months following a single administration.[6] As mentioned previously, precise gene editing can be realized through the homology-directed repair (HDR) pathway, which is the most versatile means through which nucleases can insert, delete, or replace the desired sequences at a target site using an exogenous donor DNA template.[7] The ability to perform Cas9-mediated HDR in mammalian cells has advanced the study of human genetics and enabled the construction of a wide range of cell and animal models with modifications at genomic sites of interest.[8] Zhao et al[9] used Cas9-mediated HDR to correct LdlrE208X-induced mice with familial hypercholesterolemia (FH). Although the rate of correction was as low as 6% among hepatocytes, cholesterol was reduced, and atherosclerosis phenotypes were effectively ameliorated. Recently, our team reported CRISPR/Cas9 combined with AAV-based HDR to target specific locus of in vivo cardiomyocytes, achieving an efficient HDR efficiency of up to approximately 45%. This will enable efficient and precise delivery of therapeutic transgenes to validate loci to realize precise gene therapy.[10] Base editors consist of DNA-modifying enzymes fused to a programmable DNA-targeting moiety. In 2016, Komor et al[11] reported the first cytosine base editors (CBEs), which use natural single-strand DNA deaminase domains to convert cytosine nucleotides to uracil.[12] In this manner, CBEs can convert C–G base pairs first to U–G base pairs, and ultimately to T–A base pairs following DNA repair. In 2017, Gaudelli et al[13] reported the first adenine base editors (ABEs), which convert A–T base pairs to G–C base pairs. Nuclease-impaired Cas9 (Cas9n) was fused to single-strand cytidine deaminases or deoxyadenosine to direct and limit the activity of base editors to the genomic site of interest.[14] Recently, Lan et al[15] reported the first research on ABE treatment in mice with Myh6 c.1211C >T-induced HCM, revealing great efficiency in embryos. Most recently, Olson et al[16] demonstrated a new strategy to ablate CaMKIIδ oxidation by CRISPR-Cas9 base editing for heart disease therapy. To introduce more precise DNA edits beyond those that can be achieved by base editing without requiring DSBs, Nelson et al[17] introduced prime editing (PE) in 2019. Prime editors are composed of an engineered reverse transcriptase fused to Cas9 nickase that introduces a nick in the R-loop at the target DNA site. They are able to achieve any single base-to-base change, deletions of at least 80 nucleotides, and insertions of at least 44 nucleotides. Chen et al[18] have made great efforts to improve the efficiency of PE. Although there is no published research on the use of PE to treat inherited CVDs, it is believed that the advantages of PE would help to offset the limitations of base editing. Perspectives on how to launch a practical gene therapy project in China. Although the incidence of inherited CVDs is low, the absolute number of patients with inherited CVDs in China is large given the fact that the country has the largest population in the world. Thus, providing disease management for such a population is a great challenge. To achieve this goal, we provide the following recommendations [Supplementary Figure 1, https://links.lww.com/CM9/B745]. (1) Full use of genetic testing must be made in patients suspected of having inherited CVDs to obtain an atlas of molecular diagnosis results and hotspot mutations in the Chinese population and ultimately establish a nationwide association and database to manage the results. (2) Researchers must design integrative strategies for delivering functional copies to restore the expression of impaired genes. This step includes the selection of a gene therapy method (gene replacement/addition, gene inhibition/depletion, or gene editing) and vectors. Most importantly, the gene editing protocol needs to be associated with hotspot mutations, which could be the most efficient way to achieve industrial production. (3) Preclinical validation, especially ethical approval, is critical for every single gene therapy protocol. Instruction and statements are required to direct the translation of gene therapy. (4) Potential recipients of gene therapy must be continuously and carefully followed and managed. Medical centers must join to form a cooperative organization that is able to perform clinical trials. In conclusion, preimplantation genetic testing (PGT) technology has been applied in the prevention of ICD, so that couples with genetic risk of ICD can avoid termination of pregnancy and give birth to healthy children without ICD susceptibility genes. Moreover, gene therapy is the most powerful and efficient method of managing inherited CVDs, but there is a long way to go to achieve this medical goal. At present, gene therapy shows general prospects for treating CVDs. It is believed that with further basic and clinical research, better treatment of CVDs will become a hot topic. Funding This study was supported by grants from the Key R&D Program of Sichuan Province of China (No. 2021YFQ0061), Science and Technology Department of Sichuan Province (No.2022ZYD0067 and MSGC20230024), Natural Science Foundation of China (Nos.82070324, 82001496, and 82270249), Project of Chengdu Science and Technology Bureau (No.2021-YF05-02110-SN), and China Postdoctoral Science Foundation (Nos. 2020M680149 and 2020T130087ZX). Conflicts of interest None.