Titer and product affect the distribution of gene expression after intraputaminal convection-enhanced delivery

Abstract

Background: The efficacy and safety of intracerebral gene therapy for brain disorders like Parkinson's disease depends on the appropriate distribution of gene expression.

Objectives: To assess whether the distribution of gene expression is affected by vector titer and protein type.

Methods: Four adult macaque monkeys seronegative for adeno-associated virus 5 (AAV5) received a 30-µl inoculation of a high- or a low-titer suspension of AAV5 encoding glial cell line-derived neurotrophic factor (GDNF) or green fluorescent protein (GFP) in the right and left ventral postcommissural putamen. The inoculations were conducted using convection-enhanced delivery and intraoperative MRI (IMRI).

Results: IMRI confirmed targeting and infusion cloud irradiation from the catheter tip into the surrounding area. A postmortem analysis 6 weeks after surgery revealed GFP and GDNF expression ipsilateral to the injection site that had a titer-dependent distribution. GFP and GDNF expression was also observed in fibers in the substantia nigra (SN) pars reticulata (pr), demonstrating anterograde transport. Few GFP-positive neurons were present in the SN pars compacta (pc), possibly by direct retrograde transport of the vector. GDNF was present in many neurons of the SNpc and SNpr.

Conclusions: After controlling for target and infusate volume, the intracerebral distribution of the gene product was affected by the vector titer and product biology.

Figure 1

Viral vector infusions can be identified in MRI without contrast agent. Sequential coronal brain T1 MRIs of 4 rhesus monkeys obtained after completion of CED into the ventral postcommisural putamen nucleus of a 30 μL suspension of AAV5-GFP (left hemisphere; green) and GDNF (right hemisphere; red); some backflow (fine line protruding; blue) is observed.

Figure 2

GFP was expressed mostly in neurons. High (A) and low (B) fluorescent images of the putamen nucleus injected with AAV5-GFP (GFP, green) immunostained for NeuN (red) and GFAP (purple). The white arrow shows an example of a positive neuron expressing GFP (yellow).

Figure 3

Distribution of GFP expression followed the infusion pattern and neural network. Sequential images of GFP immunostained coronal brain sections of 4 rhesus monkeys that received an inoculation of AAV5-GFP in the left ventral postcommisural putamen nucleus and one of AAV5-GDNF in the right, using convection enhanced delivery methods. Different arrows refer to positive immunostaining in needle tracks (black) or substantia nigra (white).

Figure 4

Distribution of GDNF expression followed the infusion pattern and neural network. Sequential images of GDNF immunostained coronal brain sections of 4 rhesus monkeys that received an inoculation of AAV5-GFP in the left ventral postcommisural putamen nucleus and one of AAV5-GDNF in the right, using convection enhanced delivery methods. Arrows refer to positive immunostaining in needle tracks (black) or substantia nigra (white).

Figure 5

The number of GDNF gene copies correlated with the amount of GDNF protein. (A) MRI of a coronal brain section at the level of the postcommisural putamen where brain samples were obtained for molecular analysis. (B) Pearson’s correlation graph of GDNF qPCR and ELISA data.

Figure 6

GFP and GDNF was differentially expressed in the substantia nigra. Images of the substantia nigra at the level of the emergence of the 3^rd nerve, immunostained for GFP (A, B, C) or GDNF (D, E, F) and counterstained with Nissl of a monkey that received an inoculation of AAV5-GFP in the left ventral postcommisural putamen nucleus and one of AAV5-GDNF in the right, using convection enhanced delivery methods. Top and bottom white squares in A and D correspond to high magnification images in B, C and E, F respectively. Arrows in B and C correspond to GFP negative cell bodies. Arrows in E and F correspond to GDNF positive cell bodies.

Figure 7

GDNF expression enhanced normal TH immunoreactivity. Sequential images of TH immunostained coronal brain sections of four rhesus monkeys that received an inoculation of AAV5-GFP in the left ventral postcommisural putamen nucleus and one of AAV5-GDNF in the right, using convection enhanced delivery methods. Arrows refer to the substantia nigra with increased TH immunostaining.