Pharmacokinetics (PK) in gene therapies

Pharmacokinetics for genes introduced by gene therapies is more challenging than those of finished small molecules. Small molecule drugs are administered as finished products, ready to act directly in the body. In contrast, adeno-associated virus (AAV) vectors used in gene therapies require the therapeutic agent to be synthesized within the target cell. This fundamental difference means that traditional pharmacokinetics (PK) terminology, covering absorption, distribution, metabolism, and excretion (ADME), needs to be adapted for gene therapies.

Pharmacokinetics (PK) for AAV

In the case of AAV therapies, after administration, several critical steps occur: the AAV vector binds to the target cell, the viral capsid is internalized, intracellular trafficking occurs, the capsid disassembles, the vector enters the nucleus, and the vector DNA is converted into episomal double-stranded DNA. This is followed by transcription of the transgene into mRNA and translation into the therapeutic protein.
For AAV therapeutics, PK studies focus on the biodistribution, persistence, and clearance of the vector DNA and, potentially, the transgene product. These studies aim to understand how the body processes the vector drug product, where it localizes, and how the transgene is expressed. This knowledge is crucial for determining an effective dosing strategy. The goal is to identify the lowest effective dose, minimizing side effects and manufacturing costs.

Non-clinical biodistribution or PK for gene therapies

Regulatory agencies require non-clinical biodistribution studies to assess the therapeutic product in various tissues, including blood, the injection site, kidney, liver, heart, lung, spleen, brain, spinal cord, gonads, and adrenal glands. These studies aim to:

• Evaluate the AAV vector biodistribution in target and non-target tissues.
• Correlate transgene mRNA and protein expression with efficacy and safety.
• Optimize dosing by identifying the lowest effective dose.

Clinical biodistribution studies or PK for gene therapies

In clinical settings, biodistribution studies focus on determining drug exposure and persistence in target tissues, where accessible, or in surrogate matrices. These data are critical for correlating safety and efficacy outcomes.

PK analytes: vector DNA, transgene mRNA, and protein

Pharmacokinetics for AAV therapies involves three primary analytes: vector DNA, transgene mRNA, and protein. While protein is the final therapeutic product, its levels may be below detection limits or indistinguishable from endogenous proteins. Therefore, transgene mRNA is often used as a proxy, quantified using highly sensitive RT-PCR followed by qPCR or dPCR.
We specialize in developing and validating assays for these quantifications, including extraction, RT-PCR, qPCR, and dPCR, ensuring high accuracy and sensitivity.

Pharmacodynamics and biomarker discovery

Pharmacodynamics (PD) refers to the drug’s effect on the body. For AAV vectors, the PD analyte is not the protein transcribed by the vector but the biological activity resulting from that protein’s expression. For instance:

• In hemophilia therapies, the PD analyte is the coagulation effect.
• For siRNA therapeutics, it is the gene-silencing effect.

We assess PD by identifying biomarkers that indicate the impact of transgene expression. We analyze gene expression patterns before and after treatment using RNA sequencing to discover these biomarkers. Once identified, targeted PCR assays are an option for scalability throughout drug development.

Additionally, we use Olink’s affinity-based protein panels to discover protein biomarkers from microliters of blood and plasma, enabling precise measurements even in small sample volumes.