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Gene Therapy: A Revolution in Medicine

roumen.bogoev

April 18, 2024

Editor: Roumen Bogoev

Gene therapy is a revolutionary field that has transformed the landscape of disease treatment. By harnessing the power of genes, scientists and clinicians aim to correct genetic defects, combat diseases, and enhance human health.

Gene therapy involves introducing, altering, or silencing genes within an individual’s cells to treat or prevent diseases. The main goal is to restore normal cellular function by correcting genetic abnormalities. Examples of gene editing include knocking out harmful genes, knocking in beneficial genes, and correcting harmful mutations.

Gene editing can be done ex vivo or in vivo. In ex vivo gene editing, specific cells are extracted from the patient, the genome in these cells is edited as needed and the cells are transplanted back into the patient. In in vivo gene editing, the components that perform gene editing are introduced into the patient to perform the gene-editing function “on-site” in the patient’s body. Both approaches have benefits and drawbacks. While ex vivo gene therapy involves more steps, in-vivo gene therapy is more susceptible to the damage of off-site mistargeting. The best approach for a disease may depend on the organs affected and the particular genes that need to be edited.

Figure 1. Gene therapy using ex vivo or in vivo gene editing.

The efficacy of both ex vivo and in vivo gene therapies depends on the efficiency and specificity of gene editing.

There are multiple methodologies available for gene editing, such as virus-mediated gene editing, CRISPR gene editing, Zinc finger nucleases (ZFNs), TALON, etc. Figure 2 shows the use of the CRISPR technology in editing the genomes of the cells for gene therapy (panels B and D) and viral gene editing (panel C).

Figure 2. Methods of gene editing for gene therapy. A. Cells from a patient are used to study the patient’s genome and identify areas that could benefit from gene therapy. B. The CRSIPR-Cas9 technology can be used to edit target genes in specific cells from the patient, and the edited cells are transplanted back into the patient for ex vivo gene therapy. C. AAV virus can be used as a vector to deliver genes or genetic components to the cells in the patient for in vivo gene therapy. D. CRISPR-Cas9 technology can be used for in vivo gene editing. The components needed for gene editing- the DNA sequence encoding the Cas9 enzyme and the guide RNA- are placed in a liposome-based carrier for non-viral delivery (shown here) or in a virus for viral delivery (not shown).

Virus-based methods of gene therapy

Virus-based methods for gene therapy rely on viruses such as Adenoviruses, Retroviruses, Adeno-associated viruses, the Herpes virus, etc. Many of the currently released or late-stage therapies are based on viral vectors because they are very efficient at delivering genetic material and integrating it into the genome.

Adenoviruses: Modified adenoviruses carry therapeutic genes. They efficiently infect cells but may trigger immune responses.
Retroviruses: These integrate their genetic material into the host genome. Lentiviruses, a type of retrovirus, are used for long-lasting gene expression.
Adeno-associated viruses (AAVs): Safe and effective, AAVs are widely used for gene delivery. Because of their low immunogenicity in humans, and stable and sustainable expression of exogenous genes, AAVs are often preferred for in vivo gene editing applications. The majority of the currently approved in-vivo gene therapies use AAV.
Herpes Simplex Viruses (HSV): HSV vectors are being explored for treating neurological disorders.

Figure 3. Use of viruses for gene therapy. A virus is used as a vector to house the corrected version of a target gene and introducing it back into the cell which has a defective copy of this gene.

Non-viral methods of gene therapy

Non-viral gene therapy uses delivery systems such as lipid nanoparticles (LNPs), polymeric nanoparticles, and nanoparticles with ligand modification to deliver the genes of interest into the patient's cells. Non-viral vectors may have reduced specificity1 in targeting specific organs for delivery. The ability to target certain cell types or organs is very important to minimize toxicity and side effects.

Non-viral methods use methodologies such as CRISPR-Cas9, TALEN, Zinc-finger proteins to perform gene editing. Recently mRNA has become a popular choice for function correction or editing.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a popular method due to its simplicity, efficiency, and low cost. However, it can cause off-target editing due to imperfect specificity. Further, using CRISPR-Cas9 for germline editing raises ethical questions.

TALENs (Transcription Activator-Like Effector Nucleases) exhibit high specificity with minimal off-target effects but are more time-consuming and expensive to implement than the CRISPR-Cas9 system.

ZFNs (Zinc-Finger Nucleases) can be highly specific but creating them involves intricate protein engineering which is costly and time-intensive.

mRNA (messenger RNA) molecules carry genetic instructions from DNA to ribosomes for protein synthesis. mRNA has found use for direct function correction that does not alter the genome and therefore eliminates some safety concerns that exist with viruses and other methods for implementation of gene editing. Or in some situation mRNA is used as delivery format for gene editing to better control the timing and minimize gene editing errors.

CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)

Advantages

Simplicity. With two inexpensive components- Cas9 enzymes and guide RNAs, the CRISPR-Cas9 editing system is straightforward to design and implement, making it accessible to a wide range of researchers.
Efficiency. CRISPR is highly efficient in introducing targeted mutations.
Low Cost. CRISPR is cost-effective compared to other methods.

Disadvantages

Off-Target Effects. CRISPR can occasionally modify non-targeted genes because the guide RNAs can sometimes allow DNA mismatches in a few nucleotides.
Ethical Concerns. The potential for germline editing raises ethical questions.
Limited Targeting Range. CRISPR may not work well for certain genomic regions, e.g. the highly chromatinized2 parts of the genome.

TALENs (Transcription Activator-Like Effector Nucleases)

Advantages

Simplicity. TALENs exhibit high specificity with minimal off-target effects.
Customizability. Researchers can design TALENs for specific DNA sequences.

Disadvantages

Cost and Time. TALENs are more expensive and time-consuming than CRISPR.
Efficiency. TALENs are less efficient, affecting about 1 in 140 target sequences.
Complexity. Designing TALENs requires expertise3 in protein engineering.

ZFNs (Zinc-Finger Nucleases)

Advantages

Simplicity. ZFNs can be highly specific.
Experience. ZFNs have been used for longer and have an established track record.

Disadvantages

Complex Design. Creating ZFNs involves intricate protein engineering.
Cost and Time. ZFNs are costly and time-intensive.
Limited Flexibility. Customization is more challenging³ compared to CRISPR

mRNA-Based Therapies

Advantages

Safety. mRNA methodologies do not alter the genome and therefore have fewer safety concerns.
Low Cost. mRNAs can be commercially synthesized inexpensively and at a larger scale.
Speed. In an emergency, mRNA therapeutics can be rapidly rolled out4, e.g. the mRNA-Based COVID-19 vaccines in response to the COVID-19 pandemic.

Disadvantages

Immunogenicity. While advantageous in mRNA vaccines, the activation of immune response is disadvantageous in protein replacement therapies5 when the mRNA therapy is intended to use the protein synthesized by that mRNA.

Figure 4. Gene editing using the CRSIPR Cas9 technology. In this example, a predesigned DNA fragment is being inserted into the patient’s gene. This approach can insert beneficial genes which are missing or mutated in the patient. The restoration of gene function is meant to alleviate a disease.

Breakthroughs in gene therapy

Gene therapy has achieved some remarkable breakthroughs in treating diseases. Below are a few example of the extraordinary achievements of gene therapy:

LUXTURNA^®⁶ to treat inherited retinal dystrophy

LUXTURNA is an in vivo gene therapy that can treat retinal dystrophy caused by mutations in the RPE65 gene. RPE65 gene is inherited, and individuals who inherit RPE65 mutation in both eyes can benefit from LUXTURNA. The therapy uses the AAV2 vector to deliver functional RPE65 genes in the retinal cells of those with reduced or absent biologically active RPE65 gene. The therapy consists of a one-time injection to deliver the correct RPE65 sequence to the photoreceptor cells. The treatment requires sufficient viable photoreceptor cells in the eyes that can produce the RPE65 gene after gene therapy.

ZOLGENSMA^®⁷ to treat spinal muscular atrophy in infants

ZOLGENSMA is an in vivo gene therapy that can treat spinal muscular atrophy in infants under 2 years of age. Spinal muscular atrophy occurs due to a missing or nonworking SMN1 gene. The therapy uses the AAV9 vector to deliver a functional copy of the SMN1 gene to the motor neuron cells in the infant. The one-time therapy can restore SMN1 gene function and stop progression of spinal muscular atrophy.

LYFGENIA™⁸ to treat Sickle Cell Disease

LYFGENIA is an ex vivo gene therapy that can stop vaso-occlusive events (VOEs) due to sickle cell anemia. In sickle cell anemia, there is a mutation in a gene encoding a subunit of hemoglobin. The mutated hemoglobin tends to clump together, giving the red blood cells their sickle-like shape. The sickle-shaped cells can stick to blood vessels and block blood flow. When too many blood vessels are blocked, various tissues in the body can become oxygen-deprived. This event is called a VOC.

In this therapy, blood cells are extracted from the patient and stem cells are recovered from it. Then a Lentivirus vector is used to introduce the correct and functional copy of the mutated hemoglobin gene into the patient’s blood stem cells. The cells are returned back to the body where they engraft within the bone marrow and produce new red blood cells with functional hemoglobin.

CRSIPR gene therapy and clinical trials

CRISPR shows strong promise as a method for gene editing for therapeutics. With FDA approval on December 8, 2023, Casgevy became the first FDA-approved ex vivo gene therapy that uses CRISPR-Cas9 technology. The therapy is used to treat sickle cell anemia with VOEs. The treatment uses CRISPR-Cas9 to edit patients’ hematopoietic (blood) stem cells to increase the production of fetal hemoglobin (HbF). Increased levels of HbF prevents deformation of red blood cells into the characteristic sickle shape which can reduce clumping and blocking blood vessels. Like LYFGENIA, Casgevy is an ex vivo therapy that recovers blood stem cells from the patient, makes changes to the extracted cells and then returns them back into the body to engraft with the bone marrow and produced more of the desired cell types.

As of this moment there is no FDA approved CRISPR based in vivo gene therapies. However, many new therapies using the technology are in clinical trials. A few examples are shown below:

Clinical trial for Type 1 diabetes. Developed by CRISPR Therapeutics, this therapy modifies beta cells from patients who have Type 1 diabetes so that they can make their own insulin. The trial is currently in Phase I/II⁹.
Clinical trial for transthyretin amyloidosis (ATTR amyloidosis) with cardiomyopathy. Sponsored by Intellia Therapeutics in collaboration with Regeneron Pharmaceuticals, this therapy uses CRISPR-Cas9 to reduce levels of transthyretin (TTR) protein circulating in the blood of individuals who have ATTR amyloidosis- a progressive and life-threatening disease. The trial is currently in Phase III ¹⁰.

Gene therapy- barriers to overcome

Gene therapy is a promising field that has the potential to revolutionize medicine.

However, the development is long and time consuming and some risks and challenges remain:

Unwanted immune system reaction. The body's immune system may see the newly introduced viruses as intruders and attack them. This may cause inflammation and, in severe cases, organ failure.
Targeting the wrong cells. Because gene editing methodologies can affect more than one type of cells, it is possible that they may alter other cell types and organs in the addition to the ones being targets for treatment. If this happens, healthy cells may be damaged, causing other illnesses or diseases such as cancer.
Infection caused by the virus. It is possible that once introduced into the body, the viruses may recover their original ability to cause disease.
Possibility of causing a tumor. If the new genes get inserted in the wrong spot in the patient’s DNA, there is a chance that the insertion might lead to tumor formation.

Several significant barriers still stand in the way of gene therapy becoming a widely adopted, reliable form of treatment, including:

Finding a reliable way to get genetic material into cells
Targeting the correct cells
Reducing the risk of side effects

Regardless the risks and challenges there are many new promising therapies in development.

Gene therapy reagents from GenScript

Developing a gene therapy is very challenging tasks. GenScript strives to provide support for gene therapy companies regardless of the selected methodology for gene editing. GenScript offers a complete set of services and products to meet your Gene Therapy development needs.

From early-stage discovery to preclinical and clinical development we have the tools needed for development. We also offer RUO grade, preclinical and to GMP grade quality viral and non viral gene editing solutions to meet your needs at the different stages of development or commercial production. As well as many supporting technologies utilized in the development process.

Figure 5. Wide range of services and products are available from GenScript for your gene therapy development.

References:

1) Gene Therapy and the Big Debate: Viral Vectors Vs Non-Viral Vectors

2) Li, T., Yang, Y., Qi, H. et al. CRISPR/Cas9 therapeutics: progress and prospects. Sig Transduct Target Ther 8, 36 (2023). https://doi.org/10.1038/s41392-023-01309-7

3) Pros and cons of ZNFs, TALENs, and CRISPR/Cas

4) Qin, S., Tang, X., Chen, Y. et al. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Sig Transduct Target Ther 7, 166 (2022).

5) Wei HH, Zheng L, Wang Z. mRNA therapeutics: New vaccination and beyond. Fundamental Research. 2023 Mar 16. doi: 10.1016/j.fmre.2023.02.022. Epub ahead of print. PMCID: PMC10017382.

6) Luxturna.com

7) Zolgensma.com

8) Lyfgenia.com

9) An Open-Label, FIH Study Evaluating the Safety, Tolerability, and Efficacy of VCTX211 Combination Product in Subjects With T1D - Full Text View - ClinicalTrials.gov

10) MAGNITUDE: A Phase 3 Study of NTLA-2001 in Participants With Transthyretin Amyloidosis With Cardiomyopathy (ATTR-CM) - Full Text View - ClinicalTrials.gov

LUXTURNA^® is a registered trademark of Spark Therapeutics

ZOLGENSMA^® is a registered trademark of Novartis

LYFGENIA™ is a trademark of bluebird bio, Inc.

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