Systemic gene delivery in large species for targeting spinal cord, brain, and peripheral tissues for pediatric disorders

Abstract

Adeno-associated virus type 9 (AAV9) is a powerful tool for delivering genes throughout the central nervous system (CNS) following intravenous injection. Preclinical results in pediatric models of spinal muscular atrophy (SMA) and lysosomal storage disorders provide a compelling case for advancing AAV9 to the clinic. An important translational step is to demonstrate efficient CNS targeting in large animals at various ages. In the present study, we tested systemically injected AAV9 in cynomolgus macaques, administered at birth through 3 years of age for targeting CNS and peripheral tissues. We show that AAV9 was efficient at crossing the blood-brain barrier (BBB) at all time points investigated. Transgene expression was detected primarily in glial cells throughout the brain, dorsal root ganglia neurons and motor neurons within the spinal cord, providing confidence for translation to SMA patients. Systemic injection also efficiently targeted skeletal muscle and peripheral organs. To specifically target the CNS, we explored AAV9 delivery to cerebrospinal fluid (CSF). CSF injection efficiently targeted motor neurons, and restricted gene expression to the CNS, providing an alternate delivery route and potentially lower manufacturing requirements for older, larger patients. Our findings support the use of AAV9 for gene transfer to the CNS for disorders in pediatric populations.

Figures

Figure 1

In situ hybridization of monkey spinal cords following intravenous injection of either adeno-associated virus type 9 (AAV9)-green fluorescent protein (GFP) or phosphate-buffered saline (PBS). Regions of lumbar spinal cords were probed with either antisense (a,c,e, and g) or sense (b,d,f, and h) probes against the vector-derived GFP mRNA then counterstained with fast-red as a nuclear label. Within sections incubated with antisense probe, positive labeling is shown in dark blue, and is detected in large ventral neurons (filled arrows) and glia (open arrows) throughout both the gray and white matter of all animals injected with AAV9-GFP. There was no detectable signal when vector-treated tissues were incubated with the sense probe indicating a lack of nonspecific probe binding. PBS-treated animals had no detectable signal with either the antisense or sense probes.

Figure 2

Immunofluorescent labeling of green fluorescent protein (GFP) and choline acetyl transferase (ChAT) within motor neurons. Lumbar spinal cord sections from adeno-associated virus type 9 (AAV9) or phosphate-buffered saline (PBS)-injected animals were labeled with antibodies against the vector-derived transgene (GFP; a,d,g, and j) and a motor neuron marker (ChAT; b,e,h, and k) and are shown in black and white for enhanced contrast. Merged images (c,f,i, and l), GFP in green and ChAT in red, indicate extensive transgene expression within motor neurons of the P1, P30, and P90-injected animals. GFP expression was not detected within the spinal cords of PBS-injected animals. All Bars = 200 µm.

Figure 3

Whole slide scans of adeno-associated virus type 9 (AAV9) and phosphate-buffered saline (PBS)-injected monkey brains. Representative sections through similar regions of brains from systemically injected monkey were immunolabeled with anti-GFP antibodies. Panels a,f, and k show uniform labeling throughout the sections of AAV9-injected animals but not the (p) PBS-injected animals. Boxes and arrows indicate the approximate regions from where the high magnification images were acquired. Cortex (a,b,f–j,k, and o) consistently had the highest density of GFP-expressing cells at all time points as did the lateral geniculate (a,d). Subcortical structures such as thalamus (a,c) and putamen (l,k) were well transduced but at a lower density than cortex. GFP+ cells were also seen within white matter of the pons (a,f,e) and cortex (k,m), as well as within the hippocampus (k,n). The majority of GFP+ cells in all regions had glial morphology though individual neurons could be detected throughout the brain. Brains from PBS-injected animals were negative for GFP signal (p–t).

Figure 4

Green fluorescent protein (GFP) expression within nonhuman primate skeletal muscle. GFP immunofluorescence from adeno-associated virus type 9 (AAV9)-injected and phosphate-buffered saline (PBS)-injected monkeys demonstrates extensive transgene expression in skeletal muscles of all AAV9-injected animals. GFP expression was detected in the brachial limbs (triceps brachii a–c), trunk [diaphragm e–g and transverse abdominus (TVA) i–k], pelvic limbs (quadriceps m–o, gastrocnemius q–s, and tibialis anterior u–w) and head (tongue x–z) of AAV9 systemically injected animals. No GFP signal was detected in any of the muscle from the PBS-injected animals (d,h,l,p,t, and aa).

Figure 5

Green fluorescent protein (GFP) expression within assorted organs. Of the tissues examined, GFP expression was most abundant in the (a–c) livers and (u) adrenal medulla of all AAV9-injected monkeys. Detectable GFP expression was also seen in the (e–g) kidney, (i–k) spleen, (m–o) heart, (q–s) lung, (v–x) smooth muscle of the intestines, and(z–bb) testes of AAV-injected animals. GFP was not detected in the tissues collected from PBS-injected animals (d,h,l,p,t, and y).

Figure 6

Pig spinal cord after adeno-associated virus type 9 (AAV9) injection. In situ hybridization of AAV9 (a–b) or phosphate-buffered saline (PBS) (c–d)-injected spinal cords reveals green fluorescent protein (GFP) signal within motor neurons (filled arrows) and glia (open arrows) of antisense probed sections of (a) AAV-injected pigs but not (c) PBS injected. Sense probed sections from both treated and control animals had no detectable signal (b and d). Immunohistochemical detection of GFP following (e–h) intracisternal or (i–l) intrathecal-injected spinal cords indicates extensive labeling of large ventral horn neurons within AAV9-injected (e–g and i–k) but not PBS-injected animals (h and l). Immunofluorescent colabeling of GFP (n and q) and ChAT (o and r) in spinal cord sections from intracisternal or intrathecal AAV9-injected pigs shows that the transduced cells are motor neurons (merged, m and p). C.sc, cervical spinal cord, T.sc, thoracic spinal cord; L.sc, lumbar spinal cord.

Figure 7

Adeno-associated virus type 9 (AAV9)-green fluorescent protein (GFP) mediates transgene expression in the brain after intrathecal delivery. Representative coronal brain section (a) showing GFP expression after intrathecal injection of AAV9-GFP within the hindbrain, fibers of the trigeminal nerve (b) and Purkinje cells (c). (d–f) GFP expression was also detected in ChAT+ neuron of the olivary nucleus. Cb, cerebellum; NCH, nuclei cochleares; NSV, nucleus tractus spinalis nervi trigemini; TSV, tractus spinalis nervi trigemini.