Specific Aim #4 Two key testing parameters that may significantly alter host responses

of anti-inflammatory drugs to minimize host response during initial delivery

phase.

9.2.4.1 Limitations

Given that the only difference in the follow up pilot study with the self-

complementary double stranded scAAV9.S100A1 vector was molecular, the already compiled limitations as stated in 9.2.3.1 hold for part A of this aim as well. The

performance of self-complementary vector may or may not be different if applied beyond the baseline/Post MI period. This pilot study only tested whether or not it would perform better in terms of transduction and cardiac efficacy. Despite significant changes in

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efficacy found in the IM injection group between the two vectors constructs, there was no means to determine if these events would be maintained with elevated cytokine profiles. With respect to the Part B aim of co-delivery of anti-inflammatories with AAV product, the concept was largely limited to a primitive proof of concept in vivo application. Much more detailed in vivo work would have to be performed to determine the correct drug, drug and tuned PLGA release profile that would maximize AAV therapy. The case study only featured DNA plasmid in neonatal myocytes which have different properties. 9.2.4.2 Future Direction

There are numerous parameters independent of delivery that can alter the host response profile in vivo. Follow up studies changing dose levels, promoters in the AAV constructs, co-expression of regulatory elements, and changing serotype might yield more useful in myocardial muscle in the early to late post MI periods. AAV1, AAV6 and AAV9 all have high functional reported efficacy in cardiac muscle but there have not been comparative studies assessing outcome in terms of the host response. Given that the serotype affects efficiency independent of method on a per dose basis, it is

conceivable that the more efficient vector would be less inflammatory given that dose can be lowered to reach the same target expression level. Counter to that argument, it might also be that one set of antigens of the capsid is more responsive versus others, this would be measured with ELISPOT assays at a fixed dose with the same construct. Despite the best efforts to engineer the AAV delivery vehicle to minimize expression, there is no way to stop innate immune activation following direct myocardial delivery whether it be with a needle, liquid jet or other physical method. An anti-inflammatory co- formulation strategy is novel from the sense of it being locally directed to the

myocardium rather than systemically. Numerous gene therapy trials incorporate systemic use of steroids and or immunosuppression to knockdown inflammatory host

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responses. This dissertation argues that a local blockage may actually be more effective and better for the patient’s overall health given the side effects of the current means. However, identifying the correct drug with the precise release profile must be determined to thoroughly test this strategy in future in vivo rodent study.

9.3 Conclusions

Improving clinical outcomes in present and future AAV mediated gene therapy trials for post myocardial infarction induced heart failure patients will require a systematic approach. Delivery as shown in the study series in this dissertation was proven to be a prime factor that can determine outcome along with proper dose, vector and gene selection. Delivery considerations in terms of safety and efficacy may be defined patient to patient, whereby the goal is to rescue the sufficient percentage of cells that would reach desired expression levels indicative of improved measurable outcomes. These measures must be defined within safety limits that promote host tolerance, or the cardiac gene therapy will fail. Gene therapy as a whole field is a risky enterprise due to this risk/reward complex. In the case studies where therapy fails it is most certain that there was not enough therapeutic expression or maladaptive responses mounted negating any therapeutic effect.

The inspiration of this dissertation was to advance the seemingly dead end direct delivery arm of cardiac gene delivery technology with a novel concept. The liquid jet concept was reduced to practice in a rodent model, but as stated many more technical challenges would need to be surmounted in order for it to become a viable therapeutic strategy.

In terms of market need the liquid jet technology would at minimum be suitable as an adjunctive surgical therapy with a controlled device, or perhaps a minimally

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invasive application where the transgene expression need to be localized (i.e.

angiogenesis genes). Therapeutic formulation manipulation with either altering vector molecular biology (i.e. as in our case example with double stranded vector) and co- administration with working pharmacologic compounds to address secondary problems offer solutions to manage these risks clinically. Since the goal is to treat diseased and healthy at risk myocardial areas with the least possible dose resulting in an efficacious expression profile, it is argued that delivery studies will remain a key research area to improve outcomes.

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