RNA Interference Aided Long-Term Culture of Dirofilaria immitis Third Stage Larvae

NextGenRnD® Solution No. 14

Current state-of-the-art

Canine heartworm Dirofilaria immitis

Heartworm infections represent a significant threat for dogs since parasites might interfere with organ function, typically the lungs and heart. If left untreated, heartworms live approximately 5 to 7 years and can cause the death of an animal. The canine heartworm (Dirofilaria immitis) has distinct life stages that cycle through mosquito and canine hosts. Adult D. immitis produce microfilariae typically found in the dog’s peripheral circulation. Mosquito feeding on an infected dog leads to ingestion of the microfilariae, which mature within mosquito after two molts into third-stage (L3) larvae during a two-week period. Once the mosquito deposits the L3 larvae onto the skin of an uninfected dog, larvae migrate through the bite wound, get access to subcutaneous tissue and rapidly convert to L4. Culturing Dirofilaria immitis at the L3 larvae stage is currently a roadblock to in-depth study of this transitory phase critical for the development of novel medicines and therapies. Current methodologies involve extracting L3 stage larvae from either mosquitos or dogs infected with the parasite. The isolates, however, only remain in L3 stage for 24 to 72 hours before converting to L4.

All previous studies on the inhibition of D. immitis L3→L4 stage transition result in incomplete molt. Incomplete L3→L4 molt refers to an intermediate parasite larva developmental stage, at which the L3 outer cuticle has not yet been shed, whereas beneath it the precursor of L4 cuticle (pre-L4) had already been synthesized. D. immitis genome and transcriptional signatures for various parasite developmental stages were published. This information has been exploited in this Solution by NextGenRnD to identify the critical transcripts, encoding absolutely essential components for pre-L4 cuticle synthesis and L3 cuticle degradation. Subsequently, highly specific small interfering RNAs (siRNAs) were designed to target these critical transcripts directly to prevent the synthesis of corresponding proteins and enzymes required for pre-L4 cuticle synthesis and L3 cuticle degradation. RNA Interference Aided Long-Term Culture of Dirofilaria immitis Third Stage Larvae, i.e., RNAiALTCDiL3, represents the hybrid approach exploiting RNAi technology developed specifically for D. immitis. RNAiALTCDiL3 is novel, reversible, natural, and intrinsic mechanism with a powerful amplification loop that will block the pre-L4 cuticle formation and allow to culture the L3-stage parasite for at least 30 days.

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