Michael A. Pagel

Delivery of virus-sized iron oxide particles to rodent cns neurons

Delivery of viral particles to the brain is limited by the volume of distribution that can be obtained. Additionally, there is currently no way to non-invasively monitor the distribution of virus following delivery to the central nervous system (CNS). To examine the delivery of virus-sized particles across the blood-brain barrier (BBB), dextran coated, superparamagnetic monocrystalline iron oxide particles, with a hydrodynamic diameter of 20 +/- 4 nm, were delivered to rat brain by direct intracerebral inoculation or by osmotic BBB disruption with hypertonic mannitol. Delivery of these particles was documented by magnetic resonance (MR) imaging and, unexpectedly, neuronal uptake was demonstrated by histochemical staining. Electron microscopy (EM) confirmed iron particle delivery across the capillary basement membrane and localization within CNS parenchymal cells following administration with BBB disruption. This is the first histologic and ultrastructural documentation of the delivery of particles the size of virions across the blood-brain barrier. Additionally, these dextran-coated, iron oxide particles may be useful, in and of themselves, as vectors for diagnostic and/or therapeutic interventions directed at the CNS.

Increasing volume of distribution to the brain with interstitial infusion: dose, rather than convection, might be the most important factor.

The volume of distribution in tissue (Vt) that can be achieved by direct interstitial infusion of therapeutic agents into brain is limited. The maintenance of a pressure gradient during interstitial infusion to establish fluid convection has been shown to increase the Vt of small, medium, and large molecules. We have used monocrystalline iron oxide nanocompounds, superparamagnetic particles of sizes the same order of magnitude as virions, to investigate the effect of dose, the volume of infusate, and the time of infusion on the distribution of large molecules in rodent brain. Our initial study in rats (n = 6) replicated the results of a previously described report of convection-enhanced delivery in cats. At a constant rate and concentration, the Vt increased in a linear fashion, proportional to the increases in time, volume, and dose. When using a constant rate and a constant concentration, however, it is unclear which variable or variables (dose, volume, infusion time) have the greatest influence on this effect. Therefore, we assessed each variable independently (n = 12). When the iron dose was increased from 5.3 to 26.5 micrograms, there was a three- to fivefold increase in the Vt, depending on the volume and time of infusion (2 Microliters/20 min, 24 microliters/20 min, or 24 microliters/120 min) (P < 0.001). When the volume of infusate was increased from 2 to 24 microliters, at an infusion time of 20 minutes and a dose of either 5.3 or 26.5 micrograms, there was a 43 or 52% decline in the Vt, respectively (P = 0.018). When the time for the infusion of 24 microliters was increased from 20 to 120 minutes, there was a 79% increase in the Vt at a dose of 26.5 micrograms but no change in the Vt at a dose of 5.3 micrograms. The effect associated with infusion time was not significant (P = 0.113). Magnetic resonance imaging was performed to document the distribution of monocrystalline iron oxide nanocompounds in vivo, and histochemical staining for iron was used to document the distribution of monocrystalline iron oxide nanocompounds in tissue sections. The Vt for both methods was calculated by computer image analysis, and the correlation between magnetic resonance and histological volumes was determined (r2 = 0.93). On the basis of this model, we suggest that dose, rather than convection, might be the most important variable in maximizing the Vt and improved distribution might be achieved by administering an increased concentration of agent.

Characterization of a new model of GM2-gangliosidosis (Sandhoff's disease) in Korat cats.

We have detected a disorder in Korat cats (initially imported from Thailand) that is analogous to human Sandhoffs disease. Pedigree analysis indicates that this disease in an autosomal recessive disorder in the American Korat. Postmortem studies on one affected cat showed hepatomegaly that was not reported in the only other known feline model of GM2-gangliosidosis type II. Histologic and ultra-structural evaluation revealed typical storage vacuoles. There was a marked deficiency in the activity of hexosaminidase (HEX) A and B in affected brain and liver as compared to controls. Electrophoresis of a liver extract revealed a deficiency of normal HEX A and B in the affected animals. The blocking primary enzyme immunoassay verified the presence of antigenically reactive HEX present in affected cat livers in quantities slightly elevated with respect to the normal HEX concentration in control cats. In leukocytes, obligate heterozygotes had intermediate levels of total HEX activity with a slight increase in the percent activity due to HEX A. Indeed, 4 of 11 phenotypically normal animals in addition to four obligate heterozygotes appear to be carriers using this assay. Affected brain and liver compared with control brain and liver contained a great excess of bound N-acetylneuraminic acid in the Folch upper-phase solids; thin-layer chromatography showed a marked increase in GM2-ganglioside. In summary, we have characterized the pedigree, pathology, and biochemistry of a new feline model of GM2-gangliosidosis which is similar to but different from the only other known feline model.

Gene replacement therapy in the central nervous system: Viral vector-mediated therapy of global neurodegenerative disease

This target article describes the current state of global gene replacement in the brain using viral vectors and assesses possible solutions to some of the many problems inherent in gene therapy of the central nervous system (CNS). Gene replacement therapy in the CNS is a potential means of producing a stable expression of normal human proteins in deficient cells and thus curing certain genetically inherited enzyme deficiencies and metabolic diseases as well as cancers. The two major issues to be addressed in CNS gene replacement are the delivery of genetic material to the brain and the expression of recombinant genetic material in target cells within the CNS. Focal inoculation of recombinant virions or other genetic vectors has limitations in global CNS disease. A new approach is the blood-brain barrier (BBB) disruption technique developed in this laboratory, in which hypertonic mannitol transiently shrinks the BBB endothelium, allowing passage of high molecular weight compounds and even viruses. Gene therapy of the CNS will require a viral vector system that allows long-term, nontoxic gene expression in neurons or glial cells. Retroviral vectors have limitations in CNS gene replacement, although they are suitable for expressing recombinant genes in intracerebral grafts, or toxic genes in brain tumors. Using mutant neurotropic viruses with reduced neurotoxicity (such as defective herpes simplex virus type I [HSV-1], the HSV-1 amplicon vector system we have developed, or adenovirus mutants) has potential for direct treatment of neurons. Injecting these vectors into rodent brains can lead to stable expression of foreign genetic material in postmitotic neuronal cells. We discuss our BBB disruption delivery technique, our defective HSV-1 amplicon vector system, and our feline model for the neuronal lysosomal storage disorder Gm 2 -gangliosidosis (Sandhoff disease), which may prove to be a useful model system for CNS gene therapy.

Imaging and Therapy with Rituximab Anti-CD20 Immunotherapy in an Animal Model of Central Nervous System Lymphoma

Purpose: To evaluate the effect of rituximab monoclonal antibody (mAb) on MRI tumor volumetrics and efficacy in a rat model of central nervous system (CNS) lymphoma when delivery to the brain was optimized with osmotic blood–brain barrier disruption (BBBD). Experimental Design: Female nude rats with intracerebral MC116 human B-cell lymphoma xenografts underwent baseline MRI and were randomized into 5 groups (n = 6 per group): (i) BBBD saline control; (ii) methotrexate with BBBD; (iii) rituximab with BBBD; (iv) rituximab and methotrexate with BBBD; and (v) intravenous rituximab. Tumor volumes were assessed by MRI at 1 week, and rats were followed for survival. Results: BBBD increased delivery of yttrium-90-radiolabeled mAb in the model of CNS lymphoma. Control rats showed 201 ± 102% increase in tumor volume on MRI 1 week after entering the study and median 14-day survival (range: 6–33). Tumor growth on MRI was slowed in the methotrexate treatment group, but survival time (median: 7 days; range: 5–12) was not different from controls. Among 17 evaluable rats treated with rituximab, 10 showed decreased tumor volume on MRI. All rituximab groups had increased survival compared with control, with a combined median of 43 days (range: 20–60, P < 0.001). There were no differences by route of delivery or combination with methotrexate. Conclusions: Rituximab was effective at decreasing tumor volume and improving survival in a model of CNS lymphoma and was not affected by combination with methotrexate or by BBBD. We suggest that rituximab warrants further study in human primary CNS lymphoma. Clin Cancer Res; 17(8); 2207–15. ©2011 AACR.

Delivery of Therapeutic Genes to Brain and Intracerebral Tumors

Delivery is a major issue in the treatment of neurological disease and tumors affecting the central nervous system (CNS). Delivery of genetic material to target cells at the molecular level has been reviewed elsewhere (please see the preceding chapters for reviews of new vectors and toxic genes for tumor therapy). Here we will address the macroscopic problem of delivery of vectors to brain tumors.

Abstract B38: Radiation enhances intracellular delivery of anti-MGMT oligomers to reduce protein expression in vitro and in a xenograft model

High levels of DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) in glioblastoma tumor are associated with temozolomide resistance and poor prognosis. MGMT provides a promising therapeutic target to overcome chemo-resistance and improve overall survival in cancer patients. In this study, we investigate the enhanced delivery of anti-MGMT morpholino oligonucleotide (AMON) using a sub-therapeutic dose of radiation to reduce MGMT expression of human cancer cells (T98G glioma and H460 and A549 non-small cell lung carcinoma) in vitro and in vivo. Compared to standard transfection techniques, sub-therapeutic dose of radiation enhanced intracellular AMON delivery and transiently reduced MGMT protein expression at 3 d in vitro. The optimal radiation dosage was cancer cell type dependent and ranged from 1-12 Gy. In addition, AMON delivered using sub-therapeutic dose of radiation increased cytotoxicity of T98G cells in response to temozolomide compared to radiation and temozolomide alone in vitro. In a H460 subcutaneous xenograft tumor model, intravenous AMON administration reduced MGMT protein level by 50% in irradiated tumors but not in contralateral non-irradiated lesions within the same animal. Tumor MGMT protein downregulation by AMON did not alter DNA promoter methylation status. Our results demonstrate for the first time the use of radiation to enhance the intracellular delivery of antisense oligonucleotides to modulate MGMT protein expression in vitro and in vivo. We have previously shown that antisense nucleotides can be delivered to the brain parenchyma using osmotic blood brain barrier disruption. Therefore, we believe that cancer patients with MGMT overexpression, unmethylated DNA promoter and/or resistance to DNA alkylating agents may benefit from this novel antisense therapeutic technology to improve overall survival. Citation Format: Jeffrey Wu, Prakash Ambady, DreeAnna Morris, Michael Pagel, Randy Woltjer, Joshua Walker, Leslie Muldoon, Edward Neuwelt. Radiation enhances intracellular delivery of anti-MGMT oligomers to reduce protein expression in vitro and in a xenograft model [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B38.