Long-term expression and safety of administration of AAVrh.10hCLN2 to the brain of rats and nonhuman primates for the treatment of late infantile neuronal ceroid lipofuscinosis

Abstract

Late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal, lysosomal storage disorder caused by mutations in the CLN2 gene, results in a deficiency of tripeptidyl-peptidase I (TPP-I) activity in neurons. Our prior studies showed that delivery of the human CLN2 cDNA directly to the CNS, using an adeno-associated virus serotype 2 (AAV2) vector, is safe in children with LINCL. As a second-generation strategy, we have demonstrated that AAVrh.10hCLN2, a rhesus-derived AAV vector, mediates wide distribution of TPP-I through the CNS in a murine model. This study tests the hypothesis that direct administration of AAVrh.10hCLN2 to the CNS of rats and nonhuman primates at doses scalable to humans has an acceptable safety profile and mediates significant CLN2 expression in the CNS. A dose of 10(11) genome copies (GC) was administered bilaterally to the striatum of Sprague Dawley rats with sacrifice at 7 and 90 days with no significant impact except for mild vector-related histopathological changes at the site of vector administration. A dose of 1.8×10(12) GC of AAVrh.10hCLN2 was administered to the CNS of 8 African green monkeys. The vector-treated monkeys did not differ from controls in any safety parameter except for mild to moderate white matter edema and inflammation localized to the administration sites of the vector. There were no clinical sequelae to these localized findings. TPP-I activity was >2 SD over background in 31.7±8.1% of brain at 90 days. These findings establish the dose and safety profile for human clinical studies for the treatment of LINCL with AAVrh.10hCLN2.

FIG. 1.

Histopathology of brain of AAVrh.10hCLN2- or PBS-treated rats. Rats were administered AAVrh.10hCLN2 (10¹¹ genome copies, divided into two equal, 5-μl aliquots) or PBS in the right striatum and left striatum. Top left: Schematic of where brain was sectioned. (A–D) Schematic showing location of coronal sections of the rat brains that were assessed for histopathology. (A) Section of the brain rostral to the injection site. (B) Section of the brain containing the administration site, shown as a vertical line. (C) Section of the brain caudal to the site of administration. (D) Section of the brain distal from the site of administration, containing the cerebellum and the brainstem. Areas where perivascular cuffing was observed are shown by the blue stippling in (B) and (C). (E–H) Cerebral cortex near the injection site, represented in the schematic in (B). (E) PBS, 7 days postadministration. (F) AAVrh.10hCLN2, 7 days postadministration. For (E) and (H), the arrows indicate examples of surgery-related mechanical trauma. (G) PBS, 90 days postadministration. (H) AAVrh.10hCLN2, 90 days postadministration. The inset, which is a magnification of the region indicated by the arrow, shows some signs of inflammation as shown by the presence of perivascular cuffing, predominantly with accumulation of lymphocytes and plasma cells. (I–L) Caudal cerebral cortical regions distal from the administration site. (I) PBS, 7 days postadministration; (J) AAVrh.10hCLN2, 7 days postadministration; (K) PBS, 90 days postadministration; and (L) AAVrh.10hCLN2, 90 days postadministration. No abnormal changes were noted. All histological specimens are stained with H&E. Scale bars: (E–L) 500 μm; inset in (H), 20 μm.

FIG. 2.

Anti-AAVrh.10 neutralizing antibodies evoked by CNS administration of AAVrh.10hCLN2 to nonhuman primates. Titers were assessed in serum by neutralization of in vitro gene transfer assay. Titers (average of two determinations) were evaluated presurgery (“presurgery” is defined as any time from 24 hr to 2 months before day 0, the day of surgery), and on days 7, 15, 30, and 60 postsurgery. Shown are data from treated animals killed on days 7 and 90 and the PBS control animal killed on day 7.

FIG. 3.

Behavioral evaluation before and after AAVrh.10hCLN2 administration in nonhuman primates. Nine African green monkeys were assessed for behavior (vector, n=8, of which 4 were killed at 7 days and 4 at 90 days; and PBS, n=1, which was killed at 7 days). The monkeys were videotaped for subsequent blinded behavioral analysis before surgery (pre-, defined as any time from 24 hr to 2 months before administration) and on days 7, 15, 30, 60, and 90 postadministration. The healthy sum, which is a sum of behaviors that relate to overall good health of the nonhuman primate and that includes anxiety, arousal, and quiet behavior (defined in detail in Supplementary Table S4), is plotted as a function of time. The data are shown for individual animals. The normal range for each parameter, calculated as the 5th to 95th percentile of measurement on all monkeys presurgery, is shown as the shaded area. *The PBS monkey was not evaluated at the presurgery time point. Behavior was evaluated only at the 7-day time point for this monkey.

FIG. 4.

Histopathology of the brain of AAVrh.10hCLN2- or PBS-treated nonhuman primates. Brain sections from African green monkeys were administered 1.8×10¹² genome copies of AAVrh.10hCLN2 in a total volume of 180 μl divided equally among 12 loci through 6 burr holes, or were administered PBS in the same volume and at the same locations. (A–D) PBS-administered animal killed 7 days postadministration. (A) Coronal section containing the administration site. (B–D) Higher magnification of the regions indicated by black boxes in (A): (B) site of administration; (C) region adjacent to site of administration; (D) region distant from the site of administration. (E–H) Representative AAVrh.10hCLN2-treated animal killed 7 days postadministration. (E) Coronal section containing the administration site. (F–H) Higher magnification of the regions indicated by black boxes in (E): (F) site of administration; (G) region adjacent to site of administration; (H) region distant from the site of administration. (I–L) A representative AAVrh.10hCLN2-treated animal killed 90 days postadministration. (I) Coronal section containing the administration site. (J–L) Higher magnification of the regions indicated by black boxes in (I): (J) site of administration; (K) region adjacent to site of administration; (L) region distant from the site of administration. In the 7- and 90-day vector-treated animals, various degrees of inflammatory response can be seen and includes gliosis [dashed arrows, (F) and (J)] hemorrhage [arrow, (F)] and perivascular cuffing predominantly by lymphocytes and histiocytes, which are types of tissue macrophage [dotted arrows, (J)]. No inflammatory changes were noted in regions adjacent to and distant from the administration site. All histological specimens are stained with H&E. Scale bars: (A, E, and I) 5 mm; 0.5 mm for all other panels.

FIG. 5.

Distribution of tripeptidyl-peptidase I (TPP-I) activity in the CNS of nonhuman primates after CNS administration of AAVrh.10hCLN2. African green monkeys were injected with AAVrh.10hCLN2 at a dose of 1.8×10¹² genome copies in a total of 180 μl divided equally among 12 loci through 6 burr holes, or were injected with PBS in the same volume and at the same location. After sacrifice the nonhuman primate brains were hemisected. The left hemispheres were sectioned into 1-cm³ sections (referred to as cubes); these cubes were homogenized and assayed for TPP-I activity. The activity was measured as fluorescence units per minute per milligram of protein. TPP-I activity from all the cubes in the PBS control animal was averaged and this was taken to be the endogenous background. In (A–C), the lighter cubes represent regions that have an activity of TPP-I less than 2 SD above the background, and the darker cubes represent the regions that have an activity greater than 2 SD above endogenous background levels; the data are shown as a three-dimensional representation of distribution of TPP-I activity. (A) The data from the control PBS-injected animal. (B) Data from one representative animal administered AAVrh.10hCLN2 and killed at 7 days. (C) Data from one representative animal treated with AAVrh.10hCLN2 and killed at 90 days. (D) Percentage of CNS with TPP-I activity greater than 2 SD above endogenous background is shown as a bar graph for all groups in the study (n=1 PBS killed at 7 days, n=4 AAVrh.10hCLN2 killed at 7 days, n=AAVrh.10hCLN2 killed at 90 days) and is compared with the historical data obtained from our study using AAV2hCLN2 in the same setting.