Treating Transthyretin Amyloidosis via Adeno-Associated Virus Vector Delivery of Meganucleases

Abstract

Transthyretin amyloidosis (ATTR) is a progressive and fatal disease caused by transthyretin (TTR) amyloid fibril accumulation in tissues, which disrupts organ function. As the TTR protein is primarily synthesized by the liver, liver transplantation can cure familial ATTR but is not an option for the predominant age-related wild-type ATTR. Approved treatment approaches include TTR stabilizers and an RNA-interference therapeutic, but these require regular re-administration. Gene editing could represent an effective one-time treatment. We evaluated adeno-associated virus (AAV) vector-delivered, gene-editing meganucleases to reduce TTR levels. We used engineered meganucleases targeting two different sites within the TTR gene. AAV vectors expressing TTR meganuclease transgenes were first tested in immunodeficient mice expressing the human TTR sequence delivered using an AAV vector and then against the endogenous TTR gene in rhesus macaques. Following a dose of 3 × 10¹³ genome copies per kilogram, we detected on-target editing efficiency of up to 45% insertions and deletions (indels) in the TTR genomic DNA locus and >80% indels in TTR RNA, with a concomitant decrease in serum TTR levels of >95% in macaques. The significant reduction in serum TTR levels following TTR gene editing indicates that this approach could be an effective treatment for ATTR.

Keywords: AAV; gene editing; gene therapy; meganuclease; transthyretin; transthyretin amyloidosis.

Figure 1.

Dose-dependent editing and reduction in serum TTR levels following in vivo gene editing with a meganuclease that targets both mouse and human TTR. Male Rag1^−/− mice were IV administered with 3 × 10¹³ GC/kg of AAV8.TBG.hTTR(V30M) or vehicle control on day 0 (n = 5/group). On day 14, mice were IV administered with 3 × 10¹¹, 3 × 10¹², and 3 × 10¹³ GC/kg of AAV8.TBG.M1TTR or vehicle control. Blood was collected at selected time points for serum mouse (A) and human TTR levels (B), respectively. Dashed line indicates time of meganuclease vector administration (day 14). Percentage of baseline TTR levels was calculated using the average of day 14. Mice were necropsied on day 56. (C) Liver was harvested at necropsy for evaluation of indel % in mouse gDNA, the AAV genome (AAV DNA), and human TTR RNA derived from the first vector (AAV RNA). (D) Evaluation of relative expression of mouse TTR RNA transcript. Values presented as mean ± SEM. *p < 0.05, **p < 0.001. AAV, adeno-associated virus; GC, genome copy; gDNA, genomic DNA; hTTR, human transthyretin; indel; insertion and deletion; IV, intravenous; SEM, standard error of the mean; TTR, transthyretin.

Figure 2.

Gene editing of human TTR in mice results in reduced serum human TTR levels. Male Rag1^−/− mice were IV administered 3 × 10¹³ GC/kg AAV8.TBG.hTTR(V30M) on day 0 (n = 5 per group). On day 14, mice were IV administered 3 × 10¹¹, 3 × 10¹², or 3 × 10¹³ GC/kg AAV8.TBG.M2TTR. Blood was collected at selected time points for human TTR levels (A). Dashed line indicates time of meganuclease vector administration (day 14). We calculated the percentage of baseline TTR levels using the average of day 14. Mice were necropsied on days 28 (n = 1), 42 (n = 2), and 56 (n = 2). (B) Livers were harvested at necropsy for evaluation of indel % in the AAV genome (DNA) and human TTR RNA derived from the first vector (RNA). Values presented as mean ± SEM. *p < 0.001.

Figure 3.

Translation of TTR gene editing from mice to NHPs. Rhesus macaques were IV administered 6 × 10¹² and 3 × 10¹³ GC/kg of AAV8.TBG.M1TTR or AAV8.TBG.M2TTR. We performed liver biopsies on day 18 and 128 postvector administration. Animals administered with AAV8.TBG.M1TTR were necropsied 1-year postvector administration. We extracted DNA and RNA and performed NGS analysis, including AMP-Seq on DNA (A), Amplicon-Seq on RNA (B), and ITR-Seq on DNA (C). ITR, inverted terminal repeat; NGS, next-generation sequencing; NHP, nonhuman primate.

Figure 4.

Substantial reduction in serum TTR levels in NHPs following systemic administration of AAV8.TBG.M2TTR. Rhesus macaques were IV administered 6 × 10¹² or 3 × 10¹³ GC/kg of AAV8.TBG.M2TTR. We collected blood samples at selected time points for TTR levels, which are presented as percent of baseline levels.