Real-time MR imaging of adeno-associated viral vector delivery to the primate brain

Abstract

We are developing a method for real-time magnetic resonance imaging (MRI) visualization of convection-enhanced delivery (CED) of adeno-associated viral vectors (AAV) to the primate brain. By including gadolinium-loaded liposomes (GDL) with AAV, we can track the convective movement of viral particles by continuous monitoring of distribution of surrogate GDL. In order to validate this approach, we infused two AAV (AAV1-GFP and AAV2-hAADC) into three different regions of non-human primate brain (corona radiata, putamen, and thalamus). The procedure was tolerated well by all three animals in the study. The distribution of GFP determined by immunohistochemistry in both brain regions correlated closely with distribution of GDL determined by MRI. Co-distribution was weaker with AAV2-hAADC, although in vivo PET scanning with FMT for AADC activity correlated well with immunohistochemistry of AADC. Although this is a relatively small study, it appears that AAV1 correlates better with MRI-monitored delivery than does AAV2. It seems likely that the difference in distribution may be due to differences in tissue specificity of the two serotypes.

Fig. 1

Schematic representation of the non-human primate brain in coronal section and sites of infusion. Region of corona radiata infused (light gray oval) in the right hemisphere in all three animals (NHP1, NHP2, and NHP3). Bilateral putaminal convections (dark gray ovals) were present in two NHP, whereas the remaining animal had only putaminal infusion in the right hemisphere. All three animals received thalamic infusion (black oval) in the left hemisphere.

Fig. 2

T1-weighted MR images of NHP RCD with GDL tracer. (A) Corona radiata. (B) bilateral putamen. (C) Thalamus. GDL contrast leak/reflux into subarachnoid space and along cannula tract (white arrows) is demonstrated in panel B.

Fig. 3

Coronal FMT-PET images after intracarotid MPTP. Panels A and B show a clear reduction in PET signal on the side ipsilateral to the intracarotid infusion. Bilateral RCD of putaminal targets (C and D) reveal robust bilateral GDL distribution in one subject (C), and MRI evidence of oil/air along with convection within the left putamen and otherwise robust convection within the right putamen of the other subject (D). Repeat FMT-PET after AAV2-hAADC gene therapy (E and F) shows a mild increase in bilateral PET signal in one subject (E), and marked increase in bilateral PET signal in the other animal (F).

Fig. 4

MRI correlation with histology in NHP with right corona radiata AAV1-GFP RCD. (A) T1-weighted coronal MR image of NHP brain with right corona radiata convection. GDL extends within the white matter tracts and, on this image, does not enter the adjacent head of caudate or overlying cortex. No leakage is noted in the subarachnoid space. (B) Histologic coronal brain section of animal depicted in A, showing GFP staining within the corona radiata (cr), with sparing of the corpus callosum (cc), overlying cortex, and adjacent head of caudate (Cd). (C) Low magnification view of specific GFP staining in the convected corona radiata. White matter tracts are easily seen, interspersed with positively stained nuclei. Scale-bar=0.25 mm. (D) High magnification view of boxed inset in C, showing positively stained astrocytic and oligodendrocytic nuclei. Scale-bar=0.10 mm.

Fig. 5

MRI correlation with histology in NHP with left thalamic AAV1-GFP RCD. (A) T1-weighted MR image showing cannula tract extending into left thalamus. Incomplete filling within the thalamus is noted as well as small amount of GDL leakage within the lateral ventricle (white arrow). (B) Coronal histologic section of NHP brain imaged in A, showing GFP in a pattern similar to that noted on MRI with GDL. Abbreviations: Thalamus (Th); globus pallidus (gp). (C) Low magnification view of thalamic region near cannula tip. Scale-bar=0.25 mm. (D) High magnification of boxed inset in C, showing GFP-positive cells within the thalamus. Both neurons and astrocytes are noted to be specifically stained. Scale-bar=0.10 mm.

Fig. 6

MRI correlation with histology in NHP with bilateral attempted putaminal AAV2-hAADC RCD. Panel A shows a low magnification view of the dorsolateral left head of the caudate (Cd) and overlying corpus callosum (cc). AADC staining is seen within the Cd after a medially placed cannula tract allowed it to be infused into this region rather than the left putamen. Notice the lack of specific AADC staining in the overlying corpus callosum (dotted polygon), despite GDL signal in this region on MRI (see C). Scale-bar=0.25 mm. Panel B depicts a representative view of higher magnification AADC staining in the left head of caudate. No such cellular staining was noted in the overlying white matter tracts. Scale-bar=0.10 mm. Panel C displays a coronal T1-weighted MR image of NHP brain with the bilateral RCD with GDL contrast. Although the putaminal targeting was better in the right hemisphere, there was some spread of the GDL into the internal capsule (ic) and overlying corona radiata (cr). The left-sided targeting was too medial and allowed convection of the head of the caudate and the overlying corpus callosum. Panel D shows a coronal histologic brain section of the same animal as imaged in panel C. This section is double-labeled, showing TH immunoreactivity (TH-IR) within the left striatum and paucity of TH-IR on the right, as a result of the right intracarotid MPTP infusion. The dense immunoreactivity in the left head of the caudate represents AADC IHC staining. Similar but more abundant AADC staining is seen within the substance of the right putamen, and part of the head of the right caudate. Note the absence of AADC staining within the overlying right corona radiata (dashed polygon, cr) and internal capsule, despite both of these regions showing GDL enhancement in panel C. Panel E depicts a low magnification view of the boxed region within the right putamen shown in D. Note the specific neuronal staining for AADC. Scale-bar=0.25 mm. Panel F shows a higher magnification view of the boxed region depicted within E. Notice the specific staining of medium spiny neurons. Scale-bar=0.10 mm.

Fig. 7

Vd differences between GDL, as noted on MR imaging, and specific IHC staining (for either GFP or AADC) in histologic brain sections. (A) Comparison of mean Vd of GDL, Vd of GFP (AAV1-GFP) and Vd of AADC (AAV2-hAADC) in both gray and white matter. (B) Keeping the same GDL and GFP IHC (AAV1-GFP) mean Vd comparison, we plotted the GDL mean Vd and AADC IHC (AAV2-hAADC) mean Vd in gray matter only. Error bars show SEM.