Targeting PLN-R14del cardiomyopathy in mice using DWORF gene therapy suggests the necessity of high expression levels

There is currently no successful treatment for patients with cardiomyopathy caused by the p.Arg14del mutation in the phospholamban (PLN) gene, other than heart transplantation. Interestingly, transgenic overexpression of the small peptide Dwarf open reading frame (DWORF) could delay disease development and extend life span in a PLN-R14del cardiomyopathy mouse model. Here, we evaluated the potential of DWORF gene therapy using adeno-associated virus cardiotropic serotype 9 (AAV9) in a PLN-R14del mouse model. At postnatal day 7, AAV9-cTnT-DWORF (AAV-DWORF) or control AAV9-cTnT-luciferase (AAV-luciferase) was delivered to homozygous PLN-R14del (R14 ^Δ/Δ ) mice. The AAV-DWORF construct targeted the majority of cardiomyocytes in the heart, as shown by in situ RNA labeling. Moreover, it significantly increased DWORF protein expression by fivefold, but did not reach the same high 25-fold increased expression level as observed in a DWORF transgenic mice. Previous study showed that high transgenic DWORF expression was able to delay PLN-R14del cardiac impairment and preserved cardiac ejection fraction (EF) in 6-week-old mice. However, the current study reveals that AAV-DWORF did not delay the decline in EF of R14 ^Δ/Δ mice. This was corroborated by the observation that the R14 ^Δ/Δ -induced increase of heart failure markers and cardiac fibrosis were not inhibited by AAV-DWORF. In conclusion, whereas high transgenic DWORF overexpression can delay cardiac dysfunction in a PLN-R14del mouse model, a fivefold lower AAV-mediated overexpression was not sufficient to delay disease progression. This suggests that only high supraphysiological DWORF levels are able to delay disease development, requiring further optimization of AAV-DWORF therapy.