It has been more than three decades since pioneering scientists introduced the concept of “gene therapy.” They proposed that replacing the deficient or altered gene with the healthy allele through gene addition could yield enduring therapeutic benefits for individuals affected by specific conditions, particularly monogenic diseases.
This breakthrough has recently materialized in treating certain ailments such as hemoglobinopathies, immunodeficiencies, and other monogenic disorders.
The multitude of emerging gene therapies holds the capacity to revolutionize healthcare across various therapeutic domains. In this article, we’ll highlight some recent breakthroughs in gene therapy and the potential impact it may have on reshaping the traditional approach to disease treatment.
- gRNA Design Tools
gRNA design tools play a crucial role in gene therapy by assisting in the design and selection of appropriate guide RNA (gRNA) sequences for targeted gene editing.
These tools employ various algorithms and computational methods to identify potential gRNA sequences that exhibit high specificity and efficiency in targeting the desired gene or genomic region. It takes into account factors such as off-target effects, GC content, secondary structures, and potential binding affinity to improve the accuracy and success rate of gene editing.
By utilizing the gRNA design tool, researchers and clinicians can streamline the process of selecting optimal gRNA sequences, which is essential for successful gene therapy applications. The tool assists in minimizing off-target effects, reducing unintended mutations, and increasing the precision of gene editing.
Moreover, gRNA design tools also contribute to the advancement of gene therapy by enabling the targeting of specific disease-causing genes or mutations associated with various genetic disorders. This technology holds significant potential in the development of personalized and targeted therapies for monogenic diseases, cancer, and other genetic conditions.
- Viral Vectors
Viruses have consistently proven themselves as the preferred carriers for delivering therapeutic genes to challenging cellular targets, enduring both successes and setbacks. In their natural state, viruses adeptly exploit host cells, instigating devastating disease outbreaks and global pandemics. However, their exceptional capacity to infiltrate cells and deceive the host into reproducing their own genetic material renders them invaluable tools in gene therapy.
Drawing upon millions of years of evolution, viruses possess a remarkable advantage over alternative gene therapy vectors, such as synthetic nanoparticles. Viruses are inherently predisposed to this purpose, allowing us to harness their natural mechanisms for our own benefit.
In the last decade, concerted efforts to overcome previous failures have yielded promising results, ushering in an era of optimism, particularly in the treatment of genetic eye disorders. Globally, there are nearly 300 ongoing clinical trials investigating gene therapies utilizing viral vectors as their foundation.
- Retroviruses and Lentiviruses
Retroviruses, including lentiviruses, are distinct with specific characteristics. Retroviruses are spherical enveloped RNA viruses measuring approximately 100 nm in diameter, capable of transfecting dividing cells. They share common genes encoding various proteins that determine cellular tropism. Lentiviruses, as a subset, possess additional genes enhancing viral titer and pathogenesis. Retroviruses, including lentiviruses, are preferred for ex vivo gene therapies due to their integration capability, ensuring sustained expression. However, caution is needed to minimize integrational mutagenesis.
HIV-1 is the extensively studied lentivirus used in gene therapy. Advances in generation, such as removing unnecessary virulence factors, have improved safety. Third-generation vectors further enhance safety by reducing recombinations required for replication-competent viruses. Pseudotyping with other envelope proteins expands the tropism. Vesicular stomatitis virus glycoprotein G is commonly used for broader transfection.
- Herpes Simplex Viruses
Herpes simplex viruses (HSV) are enveloped viruses with a genome length exceeding 150 kb, encoding nearly 90 genes. Approximately half of these genes are nonessential and can be removed, allowing for the insertion of large transgenes. HSV comprises eight types, all capable of infecting humans and causing diseases. HSVs exhibit broad tropism, particularly towards neurons, making them suitable for delivering larger transgenes without the risk of insertional mutagenesis. However, HSVs have the ability to establish latency, leading to sporadic reactivation and cytotoxicity. Additionally, proteins expressed by HSVs can induce inflammation and cytotoxic effects.
To address these concerns, a specific modification involving HSV amplicons has been developed. These are simplified HSV vectors lacking protein-coding genes and relying on helper viruses for replication and packaging. Nonetheless, producing and delivering amplicons at sufficiently high titers pose challenges in terms of cross-contamination and cytotoxicity. The latest generation of replication-competent HSV vectors has been designed to prevent latency and finds applications in oncolytic therapies and vaccines. These vectors are attenuated forms of the native HSV virus, retaining replication capabilities in vitro while being unable to replicate in vivo.
- Adeno-Associated Viruses
Adeno-associated viruses (AAVs) are small, non-enveloped DNA viruses belonging to the Parvoviridae family. With their icosahedral capsid and single-stranded DNA genome, AAVs measure about 25 nm in size. They possess a remarkable safety profile, wide tropism, and stable expression, making them highly desirable as in vivo vectors. The AAV genome consists of two open reading frames (REP and CAP) flanked by inverted terminal repeat (ITR) sequences, encoding for capsid proteins (VP1-3), non-structural factors aiding replication and assembly (REP), and a crucial capsid assembly protein (AAP). Different AAV serotypes, determined by binding receptors and tissue tropism, have been identified, each with its own unique characteristics.
AAV vectors can be constructed in three ways: single-stranded (ss), self-complementary (sc), or split genome (Figure 4b). The ssAAVs, by removing the viral genome except for ITR sequences, can accommodate transgenes up to 5 kb. However, they require second-strand synthesis in the nucleus for optimal transduction efficiency. The scAAVs, designed to bypass this step, have self-complementary vector backbones, enhancing transduction efficiency at the cost of reduced transgene capacity. Recent advancements have introduced the split genome approach, enabling transgene capacity of up to 15 kb by utilizing multiple AAV vectors. However, higher viral doses are necessary to achieve sufficient transgene expression.
- Non-Viral Vectors
Non-viral vector designs provide an alternative to viral vectors in gene therapy, addressing concerns related to cytotoxicity, immunogenicity, and mutagenesis. Polymers and lipids are commonly used non-viral vectors. Polymers form polyplexes that efficiently deliver genetic material into cells. Lipids, such as liposomes and lipid nanoparticles, interact with nucleic acids and enable effective gene delivery. These non-viral vectors offer promising potential for safer and more versatile gene therapies.
The field of gene therapy has made significant strides in recent years, offering promising treatments for monogenic diseases, cancer, and other genetic disorders. The development of advanced tools has facilitated precise and targeted gene editing, enhancing the success rate of gene therapy applications. Viral vectors, including adeno-associated viruses (AAVs), retroviruses, and lentiviruses, have demonstrated their suitability as efficient carriers for therapeutic genes, with AAVs standing out for their safety and versatility.
Furthermore, non-viral vectors, such as polymers and lipids, provide alternative delivery systems with reduced cytotoxicity and immunogenicity. These advancements can potentially reshape traditional approaches to disease treatment and pave the way for personalized and targeted therapies in the future.