Determination of hFIX gene transfer efficiency and distribution in liver. Monkeys that received 5 × 1012 vg/kg vector were biopsied at week 4 and humanely killed at week 25. (A) DNA FISH for hFIX (i and ii), and immunofluorescent staining for AAV-8 capsid protein (iii and iv) were performed on liver biopsies from an AAV8-injected animal (R3, i and iii) and from a noninjected control animal (R21, ii and iv). The detecting probe in DNA FISH and the detecting antibody in IF were labeled with Alexa fluor 488, so that positive signals appear as green punctuated dots (some identified by arrows in i and iii). Blue signal represents nuclei counterstained with DAPI (iii and iv). (B) Southern blotting on gDNAs isolated from individual lobes of rhesus liver collected at necropsy. LL indicates left lateral lobe; LM, left medial lobe; Q, quadrate lobe; RL, right lateral lobe; and RM, right medial lobe. Standards were generated with BamHI/BglII-digested plasmid DNA pAAV-hFIX spiked in pooled liver gDNAs from naive macaques at specified quantities. (C) Quantification of the copy numbers of transgene DNA per diploid genome in individual lobes of liver in each monkey as determined by Southern blotting analysis. Individual subjects, their preexisting AAV8 neutralizing titers, and the doses of AAV8-hFIX vector administered are as labeled.
