Fig. 1: CAP-Mac selection and characterization strategy.

a, AAV.CAP-Mac is a novel vector that enables brain-wide, systemic gene transfer in NHPs. Representative images are shown from a newborn rhesus macaque brain expressing three fluorescent reporters delivered intravenously using AAV.CAP-Mac (total dose 5 × 10¹³ vg, 4 weeks post-injection). b, Schematic of the CAP-Mac selection strategy. (1) CAP-Mac is an AAV9 variant that we selected from a library screened in the adult common marmoset. We generated diversity by introducing 7 NNK degenerate codons after Q588 in the AAV9 cap genome and produced the capsid library for in vivo selections in adult male marmosets. (2) In two rounds of selections, we intravenously administered 2 × 10¹² vg per marmoset (two marmosets per round), narrowing our variant pool with each round of selection. After the first round of selection, we recovered 33,314 unique amino acid sequences in the brain. For the second round of selection, we generated a synthetic oligo pool containing each unique variant plus a codon-modified replicate (66,628 total sequences). After the second round of selection, we constructed network graphs of high-performing variants, and selected two capsids—AAV.CAP-Mac and AAV.CAP-C2—to be included in the pool selections in newborn rhesus macaques. (3) For pool selections, we produced eight capsids packaging ssCAG-hFXN-HA, each with a unique molecular barcode in the 3′ UTR. This construct design enabled us to assess the protein expression of the pool by staining for the HA epitope and quantify the barcodes in viral DNA and whole RNA extracts. We injected 1 × 10¹⁴ vg kg⁻¹ of the virus pool into two newborn rhesus macaques via the saphenous vein and recovered tissue 4 weeks post-injection. (4) We moved forward with the individual characterization of AAV.CAP-Mac in various contexts (ex vivo, in vitro and in vivo) in multiple primate species.