Basic Science and Experimental Study
A Pharmacokinetic Analysis of Molecular Cardiac Surgery With Recirculation Mediated Delivery of βARKct Gene Therapy: Developing a Quantitative Definition of the Therapeutic Window

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Abstract

Background

Two major problems for translating gene therapy for heart failure therapy are: safe and efficient delivery and the inability to establish a relationship between vector exposure and in vivo effects. We present a pharmacokinetics (PK) analysis of molecular cardiac surgery with recirculating delivery (MCARD) of scAAV6-βARKct. MCARD’s stable cardiac specific delivery profile was exploited to determine vector exposure, half-life, and systemic clearance.

Methods and Results

Five naive sheep underwent MCARD with 1014 genome copies of scAAV6-βARKct. Blood samples were collected over the recirculation interval time of 20 minutes and evaluated with quantitative polymerase chain reaction (qPCR). C(t) curves were generated and expressed on a log scale. The exposure, half-life, and clearance curves were generated for analysis. qPCR and Western blots were used to determine biodistribution. Finally, all in vivo transduction data was plotted against MCARD’s PK to determine if a relationship existed. Vector concentrations at each time point were (cardiac and systemic, respectively): 5 minutes: 9.16 ± 0.15 and 3.21 ± 0.38; 10 minutes: 8.81 ± 0.19 and 3.62 ± 0.37; 15 minutes: 8.75 ± 0.12 and 3.69 ± 0.31; and 20 minutes: 8.66 ± 0.22 and 3.95 ± 0.26; P < .00001. The half life of the vector was 2.66 ± 0.24 minutes. PK model data revealed that only 0.61 ± 0.43% of the original dose remained in the blood after delivery, and complete clearance from the system was achieved at 1 week. A PK transfer function revealed a positive correlation between exposure and in vivo transduction. Robust βARKct expression was found in all cardiac regions with none in the liver.

Conclusion

MCARD may offer a viable method to establish a relationship between vector exposure and in vivo transduction. Using this methodology, it may be possible to address a critical need for establishing an effective therapeutic window.

Section snippets

Methods

The complete study design consisted of 3 separate parts:

  • 1.

    MCARD of gene with blood sample collection and variable monitoring.

  • 2.

    Applying a PK model to the data derived from blood samples and components.

  • 3.

    Quantitative analysis of GC biodistribution in tissues after animal sacrifices at 10 weeks.

Results

Significantly higher cardiac versus systemic [cardiac; systemic] concentrations were observed (Log GC/mL) over the time of recirculation at 5 minutes [9.16 ± 0.15; 3.21 ± 0.38], 10 minutes [8.81 ± 0.19; 3.62 ± 0.37], 15 minutes [8.75 ± 0.12; 3.69 ± 0.31], and 20 minutes [8.66 ± 0.22; 3.95 ± 0.26] (Fig. 3; P < .00001). The average initial concentration (1014 GC dose/circuit volume) was 11.55 ± 0.08 and a function of cardiac circuit start volume, which varies from subject to subject in the range

Discussion

In most cases, MCARD transfers >99% of the initial dose from the cardiac circuit to the cardiac interstitium or at the cardiac endothelial barriers, based on these 2 facts: Negligible vector remains in either compartment at the end of the 20-minute recirculation period; and the cardiac and systemic timepoints at 5, 10, 15, and 20 minutes all contain much less than 1% of initial dose (Fig. 3). Our most significant finding was that, surprisingly, most transfer occurs in the first 5 minutes of

Disclosures

None.

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Funding: National Heart Lung and Blood Institute (1-R01-HL083078-01A2).

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