Gene Therapy Assays

Viral Vectors

• Adenovirus: Can deliver DNA to both dividing and non-dividing cells but may cause immune responses.
• Adeno-associated Virus (AAV): Preferred for its low immunogenicity and ability to integrate into the host genome at specific sites.
• Lentivirus: Derived from HIV, can integrate into the host genome and is effective for long-term gene expression.
• Retrovirus: Integrates into the host genome but mainly infects dividing cells, posing a risk of insertional mutagenesis.
• Human Bocavirus (HBoV) Vectors: Effectively infects human airway epithelial cells, making it suitable for cystic fibrosis (CF) gene therapy.
• Foamy Viral Vectors (FVVs): FVVs are capable of delivering large genetic cargos.
• Simian Virus 40 (SV40) Vectors: Offer a non-immunogenic alternative to AAV vectors.
• Baculovirus Vectors: Derived from insect viruses, can package and deliver large genetic cargos.
• Herpes Simplex Virus 1 (HSV-1) Vectors: Engineered to carry large or multiple genes.
• Anellovectors These ssDNA viruses have been vectorized to evade the immune system and deliver DNA payloads to multiple tissues.
• Oncolytic Viruses: Viruses that specifically target and destroy cancer cells while leaving normal, healthy cells unharmed.

Non-Viral Methods

• Liposomes and Lipid Nanoparticles: Lipid espheres that encapsulate DNA/RNA and facilitate cellular uptake through lipid fusion with the cell membrane, endocytosis or direct translocation.
• Electroporation: Uses electrical pulses to create temporary pores in the cell membrane, allowing DNA/RNA/protein to enter.
• Gene Gun: Delivers DNA-coated gold or tungsten particles into cells via high-velocity microprojectiles.
• Nanoparticles: Uses various materials (e.g., polymers, lipids, gold) to protect and deliver genetic material to target cells.
• Cell-Penetrating Peptides: Peptide-based delivery systems are gaining attention for their ability to facilitate cellular uptake of therapeutic cargo.
• Engineered Extracellular Vesicles: Extracellular vesicles (EVs) are emerging as promising natural nanocarriers for gene therapy.
• DNA/RNA Nanostructures: Using nucleic acid-based nanostructures for gene delivery.

Gene Editing Technologies

• CRISPR-Cas9: Utilizes RNA-guided Cas9 nuclease to introduce site-specific double-strand breaks in DNA, allowing for gene correction, insertion, or deletion.
• TALENs (Transcription Activator-Like Effector Nucleases): Customizable DNA-binding proteins that induce double-strand breaks at specific sites.
• ZFNs (Zinc Finger Nucleases): Engineered proteins that create targeted double-strand breaks for gene modification.
• Bridge RNA: Synthetic RNA molecule designed to enhance the specificity, efficiency, and delivery of gene editing tools by facilitating targeted interactions between the synthetic RNA and their DNA target.
• Base editors: Induce chemical modifications in the nucleotide bases of DNA, converting one base into another, thereby correcting mutations at the genetic level with potentially fewer unintended consequences.

Antisense Oligonucleotides (ASOs)

Short, synthetic strands of nucleotides designed to bind to specific mRNA or DNA molecules, blocking their transcription or translation or modifying splicing patterns.

RNA Interference (RNAi)

Uses small interfering RNAs (siRNAs) or microRNAs (miRNAs) to degrade or inhibit the translation of target messenger RNAs (mRNA).

Ex vivo Gene Therapy

Involves removing cells from a patient, genetically modifying them in the laboratory, and then reintroducing the modified cells back into the patient. Commonly used for hematopoietic stem cells and T cells.

In vivo Gene Therapy

Directly delivers genetic material into a patient’s body, targeting specific cells or tissues.

Direct Injection

Injection of genetic material directly into tissues, commonly used in localized treatments like muscle or tumor tissues.