**4. Conclusions**

block copolymers, were first reported by Lemieux et al. [70] as efficient formulations for muscle gene delivery. Contrary to polycationic molecules, these delivery systems do not condense DNA into small particles and display no or weak interactions with nucleic acids [69,71]. Direct intramyocardial injection of poloxamer/DNA formulations showed no toxic effect towards the myocardium although gene expression remained limited and restricted to the injection site [71]. To increase the diffusion of poloxamer/DNA formulations into the myocardium, further experiments conducted *in vivo* on larger animals, through a clinically relevant administration route, were performed. As seen in **Figure 3**, this resulted in similar gene expression rate compared to that of the same transgene delivered using an AAV1 vector. To provide more insight into poloxamer-based delivery systems, further studies addressing their mechanism of action as well as experiments evaluating the possibility to readminister these formulations should be

180 In Vivo and Ex Vivo Gene Therapy for Inherited and Non-Inherited Disorders

The principal limitation of most non-clinical studies and some clinical trials was the inability to efficiently transfer genes to the cardiac ventricles. Although *in vivo* experiments using small animals may show efficient gene transfer, many fundamental differences exist between small animal and human hearts. Large animal studies are best suited for comprehensive evaluation at the preclinical stages of therapeutic development. It might seem obvious that delivery methods should meet all criteria of clinically relevant practices. Nevertheless, some preclini-

With regards to the first step to translate *in vivo* gene transfer into clinically relevant gene therapy and based at least partly on the use of naked DNA, physical methods like direct intra-myocardial injections have demonstrated feasibility, but also limited efficiency. Derived from these pioneering steps, several refinements have been introduced over time. In the context of rhythm control, one should look with interest to techniques like gene painting [72]. Gene painting refers basically to an innovative technique aimed at a very

**Figure 3.** Preclinical evaluation of intracoronary vector administration in large animals, for instance Beagle dogs. Similar amounts of cDNA were formulated differently. (A) Sample of a coronary contrast injection of the left main coronary artery in a dog heart highlighting the route of administration. (B) Mid-ventricular cross-section after AAV1 vectorization of a lacZ coding cDNA. (C) Mid-ventricular cross-section after polymer P85 vectorization of a lacZ coding cDNA. X-gal

carried out.

cal methods seem to lack this realism.

staining reveals lacZ gene expression (unpublished results).

**3.6. Administration strategies**

Gene therapy is emerging as a suitable alternative, with substantial progress in preclinical models of cardiovascular disorders. Despite the fact that none of the clinical trials, which investigated new treatments for CMs, has met their primary efficacy endpoints, subanalysis, however, has demonstrated potential efficacy. Inadequate gene delivery remains one of the underlying causes behind failures seen in clinical trials. Higher transduction efficiency is needed to achieve therapeutic effects. Use of block copolymers in gene delivery is a promising area of research, in which new and important developments are expected.

CMs can serve as a disease model for several aspects when it comes to the development of gene therapy strategies in the context of cardiac diseases, since they also engulf inherited diseases like acquired disorders.

The emphasis on gene therapies was initially focused on inherited diseases notably rescuing cardiac phenotype by introducing a functional allele in the context of recessive disorders. Even gene therapies that would only help a couple of thousand people would be a remarkable achievement.

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More recently the concept of gene therapy has been extended to a larger perspective, including the reprogramming of failing myocardial cells beyond inherited diseases. Several nonclinical studies have supported the concept, but the true challenge of gene therapy for CM remains translation into the clinic. Sticking to the old paradigm that a drug substance can only become a medicinal drug product, if one is able to formulate and administer it, it seems more obvious that gene therapy has to be clinically oriented. Treating the failing heart implies several strong constraints linked to the anatomy and physiology of the heart. Successful gene therapy approaches in other diseases support the notion, but cannot fully address the underlying specific challenges facing cardiac gene delivery.

The development of robust administration techniques and improved formulations are therefore needed before cardiac gene therapy can be integrated into the therapeutic armamentarium.
