John Bringas

Convection-Enhanced Delivery of AAV Vector in Parkinsonian Monkeys; In Vivo Detection of Gene Expression and Restoration of Dopaminergic Function Using Pro-drug Approach

Using an approach that combines gene therapy with aromatic l-amino acid decarboxylase (AADC) gene and a pro-drug (l-dopa), dopamine, the neurotransmitter involved in Parkinsons disease, can be synthesized and regulated. Striatal neurons infected with the AADC gene by an adeno-associated viral vector can convert peripheral l-dopa to dopamine and may therefore provide a buffer for unmetabolized l-dopa. This approach to treating Parkinsons disease may reduce the need for l-dopa/carbidopa, thus providing a better clinical response with fewer side effects. In addition, the imbalance in dopamine production between the nigrostriatal and mesolimbic dopaminergic systems can be corrected by using AADC gene delivery to the striatum. We have also demonstrated that a fundamental obstacle in the gene therapy approach to the central nervous system, i.e., the ability to deliver viral vectors in sufficient quantities to the whole brain, can be overcome by using convection-enhanced delivery. Finally, this study demonstrates that positron emission tomography and the AADC tracer, 6-[(18)F]fluoro-l-m-tyrosine, can be used to monitor gene therapy in vivo. Our therapeutic approach has the potential to restore dopamine production, even late in the disease process, at levels that can be maintained during continued nigrostriatal degeneration.

Distribution of Liposomes into Brain and Rat Brain Tumor Models by Convection-Enhanced Delivery Monitored with Magnetic Resonance Imaging

Although liposomes have been used as a vehicle for delivery of therapeutic agents in oncology, their efficacy in targeting brain tumors has been limited due to poor penetration through the blood-brain barrier. Because convection-enhanced delivery (CED) of liposomes may improve the therapeutic index for targeting brain tumors, we conducted a three-stage study: stage 1 established the feasibility of using in vivo magnetic resonance imaging (MRI) to confirm adequate liposomal distribution within targeted regions in normal rat brain. Liposomes colabeled with gadolinium (Gd) and a fluorescent indicator, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonic acid [DiI-DS; formally DiIC18(3)-DS], were administered by CED into striatal regions. The minimum concentration of Gd needed for monitoring, correlation of infused volume with distribution volume, clearance of infused liposome containing Gd and DiI-DS (Lip/Gd/DiI-DS), and potential local toxicity were evaluated. After determination of adequate conditions for MRI detection in normal brain, stage 2 evaluated the feasibility of in vivo MRI monitoring of liposomal distribution in C6 and 9L-2 rat glioma models. In both models, the distribution of Lip/Gd/DiI-DS covering the tumor mass was well defined and monitored with MRI. Stage 3 was designed to develop a clinically relevant treatment strategy in the 9L-2 model by infusing liposome containing Gd (Lip/Gd), prepared in the same size as Lip/Gd/DiI-DS, with Doxil, a liposomal drug of similar size used to treat several cancers. MRI detection of Lip/Gd coadministered with Doxil provided optimum CED parameters for complete coverage of 9L-2 tumors. By permitting in vivo monitoring of therapeutic distribution in brain tumors, this technique optimizes local drug delivery and may provide a basis for clinical applications in the treatment of malignant glioma.

A Review of Brain Retraction and Recommendations for Minimizing Intraoperative Brain Injury

Brain retraction is required for adequate exposure during many intracranial procedures. The incidence of contusion or infarction from overzealous brain retraction is probably 10% in cranial base procedures and 5% in intracranial aneurysm procedures. The literature on brain retraction injury is reviewed, with particular attention to the use of intermittent retraction. Intraoperative monitoring techniques--brain electrical activity, cerebral blood flow, and brain retraction pressure--are evaluated. Various intraoperative interventions--anesthetic agents, positioning, cerebrospinal fluid drainage, operative approaches involving bone resection or osteotomy, hyperventilation, induced hypotension, induced hypertension, mannitol, and nimodipine--are assessed with regard to their effects on brain retraction. Because brain retraction injury, like other forms of focal cerebral ischemia, is multifactorial in its origins, a multifaceted approach probably will be most advantageous in minimizing retraction injury. Recommendations for operative management of cases involving significant brain retraction are made. These recommendations optimize the following goals: anesthesia and metabolic depression, improvement in cerebral blood flow and calcium channel blockade, intraoperative monitoring, and operative exposure and retraction efficacy. Through a combination of judicious retraction, appropriate anesthetic and pharmacological management, and aggressive intraoperative monitoring, brain retraction should become a much less common source of morbidity in the future.

Gadolinium-loaded liposomes allow for real-time magnetic resonance imaging of convection-enhanced delivery in the primate brain

Drug delivery to brain tumors has long posed a major challenge. Convection-enhanced delivery (CED) has been developed as a drug delivery strategy to overcome this difficulty. Ideally, direct visualization of the tissue distribution of drugs infused by CED would assure successful delivery of therapeutic agents to the brain tumor while minimizing exposure of the normal brain. We previously developed a magnetic resonance imaging (MRI)-based method to visualize the distribution of liposomal agents after CED in rodent brains. In the present study, CED of liposomes was further examined in the non-human primate brain (n = 6). Liposomes containing Gadoteridol, DiI-DS, and rhodamine were infused in corona radiata, putamen nucleus, and brain stem. Volume of distribution was analyzed for all delivery locations by histology and MR imaging. Real-time MRI monitoring of liposomes containing gadolinium allowed direct visualization of a robust distribution. MRI of liposomal gadolinium was highly accurate at determining tissue distribution, as confirmed by comparison with histological results from concomitant administration of fluorescent liposomes. Linear correlation for liposomal infusions between infusion volume and distribution volume was established in all targeted locations. We conclude that an integrated strategy combining liposome/nanoparticle technology, CED, and MRI may provide new opportunities for the treatment of brain tumors. Our ability to directly monitor and to control local delivery of liposomal drugs will most likely result in greater clinical efficacy when using CED in management of patients.

Extensive distribution of liposomes in rodent brains and brain tumors following convection-enhanced delivery

Liposomes labeled with various markers were subjected to local–regional administration with either direct injection or convection-enhanced delivery (CED) into rodent brains and brain tumor models. Direct injection of liposomes containing attached or encapsulated fluorochromes and/or encapsulated gold particles indicated that tissue localization of liposomes could be sensitively and specifically detected in the central nervous system (CNS). When CED was applied, liposomes achieved extensive and efficient distribution within normal mouse brains. Co-infusion of mannitol further increased tissue penetration of liposomes. Liposomes were also loaded with gadodiamide to monitor their CNS distribution in rats by magnetic resonance imaging (MRI). CED-infused liposomes were readily seen on MRI scans as large regions of intense signal at 2 h, and more diffuse regions at 24 h. Finally, labeled liposomes were infused via CED into tumor tissue in glioma xenograft models in rodent hosts. In intracranial U-87 glioma xenografts, CED-infused liposomes had distributed throughout tumor tissue, including extension into surrounding normal tissue. Greater penetration was observed using 40 versus 90 nm liposomes, as well as with mannitol co-infusion. To our knowledge, this is the first report of CED infusion of liposomes into the CNS. We conclude that CED of liposomes in the CNS is a feasible approach, and offers a promising strategy for targeting therapeutic agents to brain tumors.

Adeno-Associated Virus Serotype 9 Transduction in the Central Nervous System of Nonhuman Primates

Widespread distribution of gene products at clinically relevant levels throughout the CNS has been challenging. Adeno-associated virus type 9 (AAV9) vector has been reported as a good candidate for intravascular gene delivery, but low levels of preexisting antibody titers against AAV in the blood abrogate cellular transduction within the CNS. In the present study we compared the effectiveness of vascular delivery and cerebrospinal fluid (CSF) delivery of AAV9 in transducing CNS tissue in nonhuman primates. Both delivery routes generated similar distribution patterns, although we observed a more robust level of transduction after CSF delivery. Consistent with previous reports administering AAV9, we found greater astrocytic than neuronal tropism via both routes, although we did find a greater magnitude of CNS transduction after CSF delivery compared with intravascular delivery. Last, we have demonstrated that delivery of AAV9 into the CSF does not shield against AAV antibodies. This has obvious implications when developing and/or implementing any clinical trial studies.

Convection-Enhanced Delivery of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand with Systemic Administration of Temozolomide Prolongs Survival in an Intracranial Glioblastoma Xenograft Model

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent activator of cell death, preferentially killing neoplastic cells over normal cells, the efficacy of TRAIL for the treatment of glioma might be limited due to cellular resistance and, importantly, poor distribution after systemic administration. TRAIL and temozolomide (TMZ) were recently shown to have a synergistic antitumor effect against U87MG glioma cells in vitro. Convection-enhanced delivery (CED) can effectively distribute TRAIL protein throughout a brain tumor mass. In this study, we evaluated CED of TRAIL, alone and in conjunction with systemic TMZ administration, for antitumor efficacy. CED of TRAIL demonstrated safe and effective distribution in both normal brain and a U87MG intracranial xenograft model. Individually, both CED of TRAIL and systemic TMZ administration prolonged survival in tumor-bearing rats. However, the combination of these two treatments was significantly more effective than either treatment alone. CED of TRAIL in conjunction with systemic TMZ treatment is a promising strategy for the treatment of malignant gliomas.

Efficient gene therapy-based method for the delivery of therapeutics to primate cortex

Transduction of the primate cortex with adeno-associated virus (AAV)-based gene therapy vectors has been challenging, because of the large size of the cortex. We report that a single infusion of AAV2 vector into thalamus results in widespread expression of transgene in the cortex through transduction of widely dispersed thalamocortical projections. This finding has important implications for the treatment of certain genetic and neurodegenerative diseases.

Eight Years of Clinical Improvement in MPTP-Lesioned Primates After Gene Therapy With AAV2-hAADC

This study completes the longest known in vivo monitoring of adeno-associated virus (AAV)-mediated gene expression in nonhuman primate (NHP) brain. Although six of the eight parkinsonian NHP originally on study have undergone postmortem analysis, as described previously, we monitored the remaining two animals for a total of 8 years. In this study, NHP received AAV2-human L-amino acid decarboxylase (hAADC) infusions into the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-lesioned putamen. Restoration of AADC activity restored normal response to levodopa and gene expression could be quantitated repeatedly over many years by 6-[(18)F]fluoro-meta-tyrosine (FMT)-positron emission tomography (PET) and confirm that AADC transgene expression remained unchanged at the 8-year point. Behavioral assessments confirmed continued, normalized response to levodopa (improvement by 35% over historical controls). Postmortem analysis showed that, although only 5.6 + or - 1% and 6.6 + or - 1% of neurons within the transduced volumes of the striatum were transduced, this still secured robust clinical improvement. Importantly, there were no signs of neuroinflammation or reactive gliosis at the 8-year point, indicative of the safety of this treatment. The present data suggest that the improvement in the L-3,4-dihydroxyphenylalanine (L-Dopa) therapeutic window brought about by AADC gene therapy is pronounced and persistent for many years.

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

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.