Cardiac-targeted delivery of regulatory RNA molecules and genes for the treatment of heart failure

Abstract

Ribonucleic acid (RNA) in its many facets of structure and function is becoming more fully understood, and, therefore, it is possible to design and use RNAs as valuable tools in molecular biology and medicine. Understanding of the role of RNAs within the cell has changed dramatically during the past few years. Therapeutic strategies based on non-coding regulatory RNAs include RNA interference (RNAi) for the silencing of specific genes, and microRNA (miRNA) modulations to alter complex gene expression patterns. Recent progress has allowed the targeting of therapeutic RNAi to the heart for the treatment of heart failure, and we discuss current strategies in this field. Owing to the peculiar biochemical properties of small RNA molecules, the actual therapeutic translation of findings in vitro or in cell cultures is more demanding than with small molecule drugs or proteins. The critical requirement for animal studies after pre-testing of RNAi tools in vitro likewise applies for miRNA modulations, which also have complex consequences for the recipient that are dependent on stability and distribution of the RNA tools. Problems in the field that are not yet fully solved are the prediction of targets and specificity of the RNA tools as well as their tissue-specific and regulatable expression. We discuss analogies and differences between regulatory RNA therapy and classical gene therapy, since recent breakthroughs in vector technology are of importance for both. Recent years have witnessed parallel progress in the fields of gene-based and regulatory RNA-based therapies that are likely to significantly expand the cardiovascular therapeutic repertoire within the next decade.

Figure 1

Traditional and current concept for coding vs. non-coding DNA functions. Only a small fraction of the RNA species found in eukaryotic cells has protein-coding function, the traditional role for RNA. During the past decade, a multitude of RNAs arising from the huge non-coding part of the genome was discovered to exert regulatory functions of fundamental importance to maintain normal cell function.

Figure 2

RNA-based technologies for gene and protein silencing. A broad spectrum of ‘tools’ made from RNA molecules has been developed that are capable of either suppressing specific gene/protein functions or of altering complex expression patterns (miRNAs). By these capacities, RNA tools significantly extend the therapeutic repertoire beyond that of classical pharmacology.

Figure 3

Current strategies for regulatory RNA modulation. The cartoon outlines the principle of RNAi alongside that of miRNA enhancement, both of which share multiple cellular components (boxes on the right), as well as strategies towards miRNA inhibition (left).

Figure 4

RNA and gene therapy—relation to other therapeutic concepts. Regulatory RNA- and gene-based strategies have recently been successfully used for cardiac therapies in vivo. Recent developments in vector technology laid the foundation for efficient, cardiac-targeted, and stable RNA and gene delivery. Parallel evolution of RNA technology enabled stable expression of RNAi-mediating small RNAs over months. A fully synthetic class of RNA molecules, spiegelmers, displays high stability in vivo and constitutes a novel class of RNA drugs with significant therapeutic potential. Only in selected cases, these fundamentally new approaches may be replaced by conventional drugs or proteins.

Figure 5

Spectrum of targets for heart failure therapy. The cartoon distinguishes two different classes of targets that need to be approached by using fundamentally distinct methods. ‘Class A’ targets (green) need to be enhanced for therapeutic purposes, whereas ‘Class B’ targets (red) require suppression. Only a fraction of the currently known targets is listed for illustration. “General” denotes therapeutic targets relevant to HF of any origin, whereas “specific” targets are relevant for specific etiologies only.

Figure 6

Protocol for RNA interference therapy of heart failure (reproduced by permission from Suckau et al.). (A) Animals for RNAi therapy were divided into two groups: one of 56 animals with aortic banding and a second of 12 sham-operated animals. In aortic-banded animals, we waited for them to develop LV dilatation and a decrease in fractional shortening by 25% before cardiac RNAi vector transfer. Of 56 aortic-banded animals, 40 survived and were divided into groups receiving AdV-shGFP, AdV-shPLB, rAAV9-shGFP, or rAAV9-shPLB. In the aortic root, 3 × 10¹⁰ pfu of each AdV was injected. For experiments with rAAV9, tail-vein injection was done with 5 × 10¹¹ genomes of either vector. Outcome evaluation by echocardiography, tip catheter, morphometry, and histology was performed after 1 month in the adenoviral and after 3 months in the rAAV9 groups (see ref.). (B) Immunohistochemical staining of GFP in different organs 1 month after intravenous injection of rAAV9-GFP. Whereas after intravenous injection of an adenoviral vector (AdV-GFP), no GFP was detected in the heart (a), rAAV9-GFP treatment resulted in strong GFP expression (b and c) which was grossly homogeneous. An average of 70% of cardiomyocytes were positive by immunohistochemistry. (e) Skeletal muscle with faint staining of a fraction of cells, whereas the liver shows prominent signal of individual cells only (d). No signal was visible in the lungs (f). Few areas were completely devoid of GFP immunoreactivity (encircled yellow areas); others showed homogeneous cytoplasmic staining (red circles). Staining was particularly dense at sites where high expression over 1 month had obviously resulted in the formation of precipitates (white arrows) of GFP. (C) Rats were injected intravenously with an rAAV9-GFP vector expressing GFP or with saline. One month later, GFP imaging showed a grossly homogeneous cardiac GFP signal in the rAAV9-GFP group (bottom) and no signal in the saline group (top). (D) Representative western blots show a significant decrease in cardiac PLB protein after 1 month of AdV-shPLB and 3 months of rAAV9-shPLB therapy, compared with shGFP control groups. The sodium–calcium exchanger and GAPDH protein remained unchanged. SERCA2a was decreased in the shGFP groups that were in HF compared with sham, whereas SERCA2a was increased significantly in both shPLB groups. *P < 0.05 compared with AdV-shGFP; ^#P < 0.05 compared with rAAV9-shGFP.