Differential Transduction Following Basal Ganglia Administration of

© The American Society of Gene & Cell Therapy

original article

Differential Transduction Following Basal Ganglia Administration of Distinct Pseudotyped AAV Capsid Serotypes in Nonhuman Primates Hemraj B Dodiya1, Tomas Bjorklund2, James Stansell III1, Ronald J Mandel3, Deniz Kirik2 and Jeffrey H Kordower1 Department of Neurological Sciences, Rush University Medical Center, Chicago, Illinois, USA; 2Brain Repair and Imaging in Neural Systems (BRAINS) Unit, Lund University, BMC D11, Lund, Sweden; 3Department of Neuroscience, University of Florida, Gainesville, Florida, USA 1

We examined the transduction efficiency of different adeno-associated virus (AAV) capsid serotypes encoding for green fluorescent protein (GFP) flanked by AAV2 inverted terminal repeats in the nonhuman primate basal ganglia as a prelude to translational studies, as well as clinical trials in patients with Parkinson’s disease (PD). Six intact young adult cynomolgus monkeys received a single 10 µl injection of AAV2/1-GFP, AAV2/5-GFP, or AAV2/8-GFP pseudotyped vectors into the caudate nucleus and putamen bilaterally in a pattern that resulted in each capsid serotype being injected into at least four striatal sites. GFP immunohistochemistry revealed excellent transduction rates for each AAV pseudotype. Stereological estimates of GFP+ cells within the striatum revealed that AAV2/5GFP transduces significantly higher number of cells than AAV2/8-GFP (P < 0.05) and there was no significant difference between AAV2/5-GFP and AAV2/1-GFP (P = 0.348). Consistent with this result, Cavalieri estimates revealed that AAV2/5-GFP resulted in a significantly larger transduction volume than AAV2/8-GFP (P < 0.05). Each pseudotype transduced striatal neurons effectively [>95% GFP+ cells colocalized neuron-specific nuclear protein (NeuN)]. The current data suggest that AAV2/5 and AAV2/1 are superior to AAV2/8 for gene delivery to the nonhuman primate striatum and therefore better candidates for therapeutic applications targeting this structure. Received 1 July 2009; accepted 18 August 2009; published online 22 September 2009. doi:10.1038/mt.2009.216

Introduction Gene therapy provides a novel means to deliver proteins to the central nervous system in a site-specific manner. Among the many different viral delivery systems, the adeno-associated virus (AAV) has become a common vector of choice to deliver genes aimed at modeling neurodegenerative diseases or delivering therapeutic molecules. AAV is a 20–25 nm nonpathogenic human parvovirus.1 Among over 100 identified serotypes of AAV capsid, AAV1–AAV10 are currently being developed as recombinant

v ectors for gene therapy applications because of their unique capsid-associated tropism for specific receptors that may confer tissue specificity. AAV vectors have a wide range of host and cell type tropism and transduce both dividing and nondividing cells in vitro and in vivo with high stability of transgene expression.2 The nonpathogenic and persistent long-term nature of AAV infection, combined with its wide range of infectivity, has not only made this virus a useful gene transfer vector but also a promising candidate vector for the gene therapy. Recombinant vectors based on AAV2 capsid proteins have been developed as a gene therapy tool and tested in preclinical trials for almost 25 years with an excellent safety profile. This serotype has been shown to transduce neurons in almost all regions of the brain.3,4 Thus, AAV2 vectors are currently being evaluated in clinical trials for central nervous system diseases.5–7 Specifically, open-label studies in Parkinson’s disease (PD) have successfully delivered the potentially therapeutic molecules using an AAV2 vector with few, if any side effects, suggesting that in vivo gene therapy in the adult human brain can be safe.8–11 Also, recent studies have shown that AAV vectors are useful to generate various animal models for neurodegenerative diseases in rodents and nonhuman primates.12–17 Due to variation in the amino-acid composition of the capsid protein(s), various serotypes differ in which receptors they utilize for cell entry and the epitopes recognized by the immune system. By simply changing the serotypes, the transduction efficiency of certain cell types may be significantly improved and may preferentially escape from immune surveillance. Several of these serotypes have been used in in vivo central nervous system transgene studies preclinically.18–24 Although AAV2 has been used in most studies including the clinical trials, other serotypes have the potential to provide improved transduction efficiency and spread of the vector from the injection sites.19,23 In addition, neutralizing antibodies against wild-type AAV2 have been detected in a significant portion of the human population that may encumbrance the application of AAV2 vector in clinical gene therapy applications.25–27 Specifically, AAV1 and AAV5 serotypes have more favorable properties for reproducible production at high titers. Specific identified cell tropism, high transduction efficiency, and low immune response are the major criteria for the

Correspondence: Jeffrey H Kordower, Department of Neurological Sciences, Rush University Medical Center, 1735 West Harrison Street, Chicago, Illinois 60612, USA. E-mail: [email protected] Molecular Therapy vol. 18 no. 3, 579–587 mar. 2010

579


Serotype Transduction in Nonhuman Primates

AAV serotype to be considered as the future gene therapy vector. The present study evaluates the transduction efficiency and cell tropism as well as safety of AAV serotypes AAV2/1, AAV2/5, and AAV2/8 in the basal ganglia of nonhuman primates. We now present data showing that AAV1 and AAV5 serotypes yield high transduction rates and neuronal tropism in the primate brain leading to the serious consideration as a future gene therapy vectors to treat neurodegenerative disorders like PD.

Results Transduction pattern of different pseudotyped AAV (AAV2/1, AAV2/5, and AAV2/8) after vector injection into the primate caudate and putamen Green fluorescent protein (GFP) immunohistochemistry revealed robust GFP expression in the cell bodies and fibers in the striatal AAV2/1-GFP

regions for all AAV pseudotypes. At 3 months after AAV injection, numerous GFP positive (GFP+) neurons were observed around the injection site (Figures 1 and 2). To quantitatively compare the transduction efficiency of each serotype, optical fractionator–based stereological cell counts were performed. The data from the unbiased estimation of the GFP+ cell population in caudate and putamen regions, shown in the Figure 3a, revealed 434,020 ± 56,173, 569,341 ± 143,415, and 222,199 ± 27,049 GFP+ cells (mean ± SEM) from the injection of AAV-GFP pseudotypes 2/1, 2/5, and 2/8, respectively. In general, all three pseudotypes provide excellent transduction with high numbers of labeled cells. AAV2/5 transduced more striatal cells than rAAV2/8 (one-way analysis of variance: F(2, 21) = 3.604; P < 0.05 post hoc Bonferroni; P < 0.04). In addition, there was no significant difference between the estimated number of cells transduced by AAV2/5 and AAV2/1

AAV2/5-GFP

a

AAV2/8-GFP

c

e

d

f

GFP

b

Figure 1 GFP expression in the caudate nucleus after AAV2/1, AAV2/5, or AAV2/8 injection. Low- and high-power photomicrographs through the caudate nucleus of GFP+ delivered via (a,b) AAV2/1, (c,d) AAV2/5, and (e,f) AAV2/8 vectors demonstrating the robust transduction of striatal cells for each serotype. Note the intense neuronal and fiber network for all pseudotypes, although the fiber network was slightly diminished with AAV2/5. Bar in e and f represents the magnifications of 1 mm and 100 µm, and applies to a,c,e and b,d,f, respectively. AAV, adeno-associated virus; GFP, green fluorescent protein. AAV2/1-GFP

AAV2/5-GFP

a

AAV2/8-GFP

c

e

d

f

GFP

b

Figure 2 GFP expression in the putamen after AAV2/1, AAV2/5, or AAV2/8 injection. Low- and high-power photomicrographs through the putamen of GFP+ delivered via (a,b) AAV2/1, (c,d) AAV2/5, and (e,f) AAV2/8 vectors demonstrating the robust transduction of striatal cells for each pseudotype. Bar in e and f represents the magnifications of 2 mm and 100 µm, and applies to a,c,e and b,d,f, respectively. AAV, adeno-associated virus; GFP, green fluorescent protein.

580

www.moleculartherapy.org vol. 18 no. 3 mar. 2010


a

b

10


AAV2/1-GFP

20

AAV2/8-GFP

a * 6

4

Transduced volume (mm3)

Transfected cells × 105

8

15

* 10

GFP

5

2

0

b AAV 1 AAV 5 AAV 8 AAV serotypes

0

d

AAV 1 AAV 5 AAV 8 AAV serotypes

Figure 3 Quantification of GFP+ cells and their distribution volume around the injection sites in caudate and putamen. (a) Stereological estimates of the number of GFP+ stained cells 3 months following intrastriatal AAV2/1-GFP, AAV2/5-GFP, and AAV2/8-GFP. One-way analysis of variance F(2, 21) = 3.604, P = 0.047, followed by Bonferroni post hoc test. *P = 0.047. (b) Quantification of GFP+ stained cell volume in the striatum 3 months following intrastriatal AAV1-GFP, AAV5-GFP, and AAV-8 GFP administration. *P < 0.05. AAV, adeno-associated virus; GFP, green fluorescent protein.

(P = 0.348). There was no significant difference seen between AAV2/1 and AAV2/8 (P > 0.05).

Transduction pattern after injection of AAV2/1 and AAV2/8 in the substantia nigra GFP immunohistochemistry showed widespread and intense GFP expression throughout the substantia nigra (Figure 4). Qualitatively, it appeared that cells within the pars compacta and pars reticulata regions were transduced with equal efficiency. Microscopic analysis revealed comparable transduction rates for both AAV2/1 and AAV2/8 serotypes. Stereological counting revealed that AAV2/1 and AAV2/8 transduced 83,402 ± 17,809 and 68,826 ± 17,501 nigral cells, respectively (no group differences, P > 0.05). The spread of AAV-GFP in the striatum To estimate the three-dimensional distribution of transgene by each serotype, the volume of individual caudate and putamen transduced for GFP+ cells was quantified. The mean volume for GFP+ cells from a single 10 µl AAV2/1-GFP injection resulted (Figure 3b) 11.6 ± 1.4 mm3. This value was similar to what was observed with a similarly titered injection of AAV2/5-GFP of a similar volume 13.2 ± 2.2 mm3. Although AAV8 also transduced a large area in caudate and putamen with a mean value of 6.4 ± 0.6 mm3, the value was significantly less than what was seen with AAV2/1 and AAV2/5 (P < 0.05 for each). Characterization of cell types transduced by different AAV pseudotypes One of our overarching goals was to understand the different tropism for the different AAV pseudotypes in the nonhuman primate brain (Figures 5–7). Confocal microscopic analyses revealed extensive colocalization of GFP with the neuronal marker NeuN for each serotype (Figure 5). In contrast, only rare GFAP+ astrocytes express GFP after AAV injections regardless of the Molecular Therapy vol. 18 no. 3 mar. 2010

c

*

Figure 4 GFP expression in nigra after intranigral AAV2/1 or AAV2/8 injections. Robust expression of GFP+ in the substantia nigra 3 months following (a,b) AAV1-GFP and (c,d) AAV8-GFP. Bar in c and d represents the magnifications of 1 mm and 50 µm. AAV, adeno-associated virus; GFP, green fluorescent protein.

pseudotype employed (Figure 6). The qualitative observations were supported by the quantitative evidence. AAV2/1, AAV2/5, and AAV2/8 demonstrated 97, 98, and 95% colocalization in neurons but 1.3, 1.5, and 2.7% in astrocytes, respectively (P > 0.05).

Immune response to each serotype LN3 immunostaining was performed to evaluate the immune response for each capsid serotype (Figure 8). LN3 stains activated T cells and monocytes as well as macrophages. In all cases, some LN3+ was observed in the area near the injection tract and partially spread to a region surrounding that tract (Figure 8b). In some cases, there appeared to be some perivascular cuffing. In no case was the staining equivalent to the volume of staining seen with GFP (Figure 8a,c,d). In this regard, some areas with abundant number of GFP+ cells displayed almost no inflammatory response (Figure 8c,d). In general, any immune response appeared to be mild. Interestingly, the LN3+ microglial infiltration was distinct from the astrogliosis that was seen as a result of the needle tract. Under these conditions, a distinct band of glial fibrillary acidic protein (GFAP) positivity was observed that followed the path of the needle tract and did not extend into the region of GFP+ (Figure 8e–g). AAV-GFP vector trafficking We did not find any GFP-ir perikarya in the substantia nigra in monkeys receiving only intrastriatal AAV (data not shown) indicating that there was no retrograde transport of the AAV or the transgene, and there was no retrograde transport for any serotype from the striatum to the cerebral cortex or thalamus as well. Although we observed fibers stained for GFP in external globus pallidus, internal globus pallidus, and substantia nigra pars reticulata indicative of anterograde transport, the experimental design of this study did not permit segregation of this finding for the different serotypes. 581



AAV2/1-GFP

AAV2/5-GFP

a

AAV2/8-GFP

g

d

GFP

GFP

e

b

NeuN

c

GFP

h

NeuN

f

Merge

NeuN

i

Merge

Merge

Figure 5 Transduction of neuronal cells by AAV pseudotypes. Confocal images (a,d,g) shows GFP+, cells (green color), extensively colocalized with (b,e,h) NeuN (red color) in the striatum as demonstrated by (c,f,i) the merged image (the yellow cells). Bar in (i) represents 100 µm for all panels. AAV, adeno-associated virus; GFP, green fluorescence protein.

Discussion At present, different AAV serotypes have generated significant interest because of their excellent transgene expression and distinct tropism.28,29 Among all identified AAV serotypes, AAV1, AAV5, and AAV8 are of particular interest because of their favorable properties of transduction patterns in the basal ganglia. The present study compared the transduction efficiency of the most efficient AAV serotypes AAV1, AAV5, and AAV8 in the nonhuman primate brains. Our results confirm and extend a previously described tropism for AAV1, AAV5, and AAV8 in rodent studies. Overall, these serotypes were shown to transduce cells in the basal ganglia with high efficiency. A primary goal of this study was to compare the transduction efficiency of AAV1, AAV5, and AAV8. As the striatum is a target of interest in many gene therapy strategies for both PD and Huntington’s disease, it was chosen as the primary injection region in this study. Furthermore, it is a well-delineated and well-characterized structure that is difficult to cover by single injections of vector in larger animal species and humans.23 By unbiased stereological estimates of GFP+ cells, we found that AAV2/1 and AAV2/5 transduce significantly higher number of cells compared to AAV2/8, whereas there was no significant difference between AAV2/1 and AAV2/5. With a single 10 µl injection, over 500,000 cells expressed GFP 3 months post-transduction. Interestingly, the Cavalieri’s volume estimates showed the same pattern of results in the distribution of AAV2/1, AAV2/5, and AAV2/8. 582

There was significant difference between AAV2/5, AAV2/1 compared to AAV2/8, whereas there was no significant difference between AAV2/1 and AAV2/5. We quantified the GFP transduced cell density in caudate and putamen; the mean value was 16,164 ± 1,830, 15,942 ± 1,486, and 11,633 ± 1,704 cells/mm3, for AAV2/1, AAV2/5, and AAV2/8, with no significant difference among the group (P = 0.115). Thus, an augmented number of successfully transduced cells seen with the AAV2/5 serotype appears to be due to the larger volume of spread and not a higher density of cells within an infected area. Confocal microscopic analyses revealed each vector displayed high neurotropism with very poor gliotropism. In this regard, >95% of the cells successfully transduced colocalized the specific neuronal marker NeuN, and it was only the rare GFP+ cell (