Gene Therapies

Author|Asteria

Editor|Hecate Ye /Introduction/

Gene therapy is a field of medical research that aims to treat disorders through genetic modification. It involves transferring genetic materials into the cells of patients. In recent years, gene therapy has made significant progress and has been approved for clinical use to treat diseases such as cancer and neurodegenerative disorders.

/Strategies/

DNA editing

One way to manipulate gene therapy is through DNA editing. There are three main DNA-editing nucleases: zinc-finger nucleases(ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-associated nucleases.

ZNFs have two domains: a DNA binding domain that recognizes the DNA in the host genome and a DNA-cleaving domain of Fok1, which is functional as a dimer and cleaves the DNA near the recognition sites. TALENs have Fok1 DNA-cleavage domains like ZNF and transcription activator-like effectors (TALEs)， which are proteins that bind to DNA and manipulate its expression. 

CRISPR systems use a guide RNA(gRNA) to target a Cas9 protein to target specific sites. gRNAs contain two parts: CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA). crRNA contains sequences complementary to the DNA sequence that is to be modified and guides Cas9 to the desired location while tracrRNA provides stability. Cas9 modifies DNA by generating double-stranded breaks(DSBs), which are repaired by nonhomologous end-joining (NHEJ). NHEJ is an error-prone DNA repair mechanism that may result in sequence insertions and deletions (INDELs). INDELs typically cause premature termination codons (PTCs), meaning that the protein synthesis process stopped earlier than expected. PTCs would be degraded by nonsense-mediated decay (NMD), a mechanism that recognizes and eliminates mRNAs containing PTCs, thus knocking out the targeted gene.

RNA editing

RNA-based editing alters RNA, which is transient, resulting in less risk of permanent side effects. The downside of this strategy is that it needs to be injected repeatedly. Antisense oligonucleotides (ASOs), which are synthetic strands of nucleic acids that interfere with the maturation of pre-mRNA to RNA through Watson-Crick base pairing, are used. During maturation, splicing, which is the process in which introns are excluded and exons are joined together, occurs. The exonic enhancers promote exon inclusion and exonic silencers promote exon exclusion. ASOs interfere with this process by including exons that are not usually translated, known as splice inclusion, or the opposite, known as splice exclusion or exon skipping.

Cas13d is another tool for RNA editing. It can be packed into a single adeno-associated virus (AAV) for delivery. AAVs are the main non-integrating vectors for in vivo gene therapy, meaning that they deliver the gene into the host cell directly without integrating the DNA into the cell. Cas13 has spacer sequences that are complementary to RNA, and it binds to target RNA, breaking it into small parts and resulting in gene knockdown. 

/Application/

Alzheimer’s Disease（AD）

Alzheimer’s disease is a neurodegenerative disorder characterized by memory loss, difficulties in communication, and impairment of daily functioning. Three genes are targeted for gene-based therapies of AD: APP and microtubule-associated protein tau (MAPT).

MAPT encodes tau proteins, which form neurofibrillary tangles in brains, causing symptoms of AD. ASOs decrease MAPT expression and thus prevent tau phosphorylation, thus lowering the levels of tau proteins in cells. APPs are glycoproteins that are cleaved by β-secretases and γ-secretases, proteolytic enzymes in brains, to produce Aβ peptides. One hallmark of AD is the accumulation of plaques, which are primarily made of amyloid β（Aβ）peptides, in brains.

Parkinson’s Disease（PD）

PD is a neurodegenerative disorder that affects movement control. It has the symptoms of rigidity, tremor, and hypokinesia, which is a decrease in physical activities. One cause of PD is the loss of dopaminergic neurons, neurons that produce and release the neurotransmitter dopamine. Gene therapies have been employed to upregulate dopaminergic signaling in PD. The dopamine synthesis pathway is regulated by three rate-limiting enzymes—GTP cyclohydrolase 1 (GCH1), tyrosine hydroxylase (TH), and aromatic amino acid DOPA decarboxylase (AADC). Gene transfer of GCH1, TH, and AADC into cells enables a high level of conversion of Levodopa precursor of dopamine and medication used in the treatment of PD, into dopamine This therapy is AAV-based and has achieved a modest relief in the symptoms.

Disease genes can also serve as gene targets. SMCA, a gene encoding α‐synuclein, acts as a gene target, as mutations in SNCA result in increased levels of α‐synuclein. In rodent models of PD, the knockdown of α‐synuclein through ASO prevents neurodegeneration, and CRISPR-mediated downregulation of α-synuclein has also shown a positive effect. LRRK2 is another disease gene. Variants in LRRK2 increase the risk of developing neurodegenerative disorders, so inhibition of LLRK2 is a viable approach for therapy. However, LLRK2 is also expressed in the lungs, kidneys, and spleen, so global inhibition of LRRK2 is risky. Gene therapy is useful as it specifically inhibits LRRK2 in brains. Intracerebral injection of LRRK2 ASOs is effective in decreasing LRRK2 mRNA levels without obvious side effects.

Chimeric antigen receptor (CAR)-T cell therapy

CAR-T therapy is a type of immune therapy that uses the power of the patient’s immune system against cancer cells. This therapy involves genetically modifying T cells to express CARs, which are synthetic receptors that are added to T cells to enhance their ability to recognize and attack cancer cells. The first step of CAR-T therapy is to collect the T cells from the patient through blood, then genetically modify the cells to express CAR, forming CAR-T cells. These cells are then cultured and expand in numbers before they are infused into the patient’s body, where they travel and seek out cancer cells. When CARs on the surface of CAR-T cells recognize the antigen in the cancer cells, they attack and destroy the cancer cells.

CARs consist of four main domains. The first domain, the extracellular target antigen-binding domain, binds to a particular antigen on the target cell. It consists of variable heavy (VH) and light (VL) chains, linked together by a linker to form a single-chain variable fragment (scFv). The second domain, the hinge region, is a flexible linker that provides stability and allows the scFv domain to bind to the targeted antigen. The length and composition of the hinge region have an impact on CAR functionality. The third domain, the transmembrane domain, anchors CAR to the T cell membrane, and it’s often derived from CD28 or CD8, which are glycoproteins found on the surface of cytotoxic T cells. It ensures that CAR inserts into T cells properly, allowing the extracellular domain to face outward and interact with the target antigen. The fourth domain is the intracellular signaling domain, which is responsible for transmitting signals and activating immune responses when the target antigen is recognized. Most intracellular signaling domain contains proteins called CD3ζ. CD3ζ contains three immunoreceptor tyrosine-based activation motifs (ITAMs), which release signals when it is phosphorylated, activating T cells.

/Conclusion/

Though gene therapies are still in the early stages and face several challenges, they provide great hope for countless patients demonstrate huge potential, and hold immense promise for the future of medicine. We hope that more research can be done so that the therapies can be used effectively and can be used to treat more patients.