Noninvasive, Targeted, and Non-Viral Ultrasound-Mediated GDNF-Plasmid Delivery for Treatment of Parkinson’s Disease


A growth factor called Glial cell line-derived neurotrophic factor (GDNF) has the remarkable ability to supports the growth and survival of dopamine-using neurons. Dopamine-using neurons are the cells that die off in Parkinson’s disease (PD). Providing GDNF to dopamine-using neurons can help them survive , but getting GDNF genes into the central nervous system relies on invasive intracerebral injections in order to pass through the blood-brain barrier.

Typically, genes are placed into the central nervous system by means of genetically engineered viruses. Viruses, however, are often recognized by the immune system and are destroyed before they can deliver their genetic payload. Therefore, non-viral gene delivery that can pass through the blood-brain barrier is an attractive alternative, since it is non-invasive. Unfortunately, such a high-yield technique is not yet available.

A new study by workers in the laboratories of Hao-Li Liu from Chang Gung University and Chih-Kuang Yeh from National Tsing Hua University, Taiwan has utilized a novel, non-viral gene delivery system to deliver genes into the central nervous system.

In this study, Lui and Yeh and their research teams used tiny bubbles made from positively-charged molecules to carry genes across the blood brain barrier. These bubbles formed stable complexes with GDNF genes, and when the skulls of laboratory animals were exposed to focused ultrasound, the bubble-gene complexes permeated the blood brain barrier and induced local GDNF expression.

(A) Schematic of GDNFp-cMBs and mechanism for controlled gene transfection of GDNFp-cMBs into brain triggered by FUS. (B) Left: Microscope bright-field images; middle: TEM images; right: PI staining image of cMBs and GDNFp-cMBs. (C) Size distributions of cMBs, GDNFp-cMBs and nMBs. (D) Zeta potential of nMB and cMB before and after adding GDNFp. (E) DNA loading efficiency of GDNFp onto nMB and cMB. The left axis was the amount of GDNFp bound onto MBs (solid line). The right axis was the GDNFp loaded efficiency onto MBs (dotted line). Single asterisk, p < 0.05, versus nMBs. Data were analyzes by Student’s paired t-test presented as mean ± SEM (n = 6 per group).
(A) Schematic of GDNFp-cMBs and mechanism for controlled gene transfection of GDNFp-cMBs into brain triggered by FUS. (B) Left: Microscope bright-field images; middle: TEM images; right: PI staining image of cMBs and GDNFp-cMBs. (C) Size distributions of cMBs, GDNFp-cMBs and nMBs. (D) Zeta potential of nMB and cMB before and after adding GDNFp. (E) DNA loading efficiency of GDNFp onto nMB and cMB. The left axis was the amount of GDNFp bound onto MBs (solid line). The right axis was the GDNFp loaded efficiency onto MBs (dotted line). Single asterisk, p < 0.05, versus nMBs. Data were analyzes by Student’s paired t-test presented as mean ± SEM (n = 6 per group).

In fact, this technique outperformed intracerebral injection in terms of targeted GDNF delivery. The amount of GDNF expressed in these laboratory animals that received the GDNF gene/microbubbles + ultrasound protocol was significantly higher than those animals that had genes directly injected into their brains. Furthermore, these higher levels of GDNF genes increased the levels of neuroprotection from PD. Animals that had a form of PD and had received nonviral GDNF gene therapy showed reduced disease progression and restored behavioral function.

This interesting study explores the potential of using ultrasound-induced passage through the blood brain barrier to bring genes into the central nervous system. This noninvasive technique successfully delivered genes into the brain to delay the effects of, and possibly treat, a neurodegenerative disease.

This study was published in Scientific Reports 6, Article number: 19579 (2016), doi:10.1038/srep19579.

 

Porous Material Helps Deliver Molecules to Stem Cell-Derived Cells


A Swedish group has successfully tested a new porous material that allows for the efficient delivery of key molecules to transplanted cells that have been derived from stem cells. Such a material can dramatically improve the way stem cell-based treatments for neurodegenerative diseases.

This research project included a collaboration between Danish, Swedish and Japanese laboratories, and it tested a new type of porous material that efficiently delivers key molecules to transplanted cells derived from stem cells in an animal model.

Mesoporous silica loaded with differentiation factors induce motor neuron differentiation in vitro. (A): Top: Scanning and transmission (inset) electron micrographs of Meso. Scale bars = 200 nm (main panel) and 50 nm (inset). Bottom: CNTF with the Cintrofin motif shown in magenta and GDNF with the Gliafin motif shown in magenta. Amino acid residues are numbered according to UniProtKB entry nos. P26441 (Cintrofin) and Q07731 (Gliafin). (B): Differentiating motor neurons (MNs) extended numerous bTUB-labeled neurites (red) on poly-D-lysine (PDL)/laminin-coated coverslips after direct administration of CNTF and GDNF or treatment with MesoMim. Neurite formation was absent from MN precursors exposed to unloaded Meso. Scale bar = 75 μm. (C): Quantitative analysis of neurite length from MNs on PDL/laminin-coated coverslips after direct administration of CNTF and GDNF, treatment with MesoMim, or treatment with unloaded Meso. Results from 7–10 experiments are expressed as mean ± SEM, and the MesoMim group is set at 100%. Direct and MesoMim administration of the factors induced a significantly greater extent of neurite outgrowth compared with the unloaded Meso group; ***, p  .05). (D): HB9-GFP+ MNs expressed the MN markers ChAT and Isl1 in a 3-day differentiation assay after treatment with CNTF and GDNF or MesoMim but not in the absence of factors. Scale bar = 25 μm. (E, F): Almost all GFP+ cells expressed Isl1 (E) and ChAT (F) after treatment with CNTF and GDNF or MesoMim. Abbreviations: bTUB, β-tubulin; ChAT, choline acetyltransferase; CNTF, ciliary neurotrophic factor; D, day; GDNF, glial cell line-derived neurotrophic factor; GFP, green fluorescent protein; Isl1, Islet 1; Meso, mesoporous silica; MesoMim, mesoporous silica loaded with peptide mimetics; Rel., relative.
Mesoporous silica loaded with differentiation factors induce motor neuron differentiation in vitro. (A): Top: Scanning and transmission (inset) electron micrographs of Meso. Scale bars = 200 nm (main panel) and 50 nm (inset). Bottom: CNTF with the Cintrofin motif shown in magenta and GDNF with the Gliafin motif shown in magenta. Amino acid residues are numbered according to UniProtKB entry nos. P26441 (Cintrofin) and Q07731 (Gliafin). (B): Differentiating motor neurons (MNs) extended numerous bTUB-labeled neurites (red) on poly-D-lysine (PDL)/laminin-coated coverslips after direct administration of CNTF and GDNF or treatment with MesoMim. Neurite formation was absent from MN precursors exposed to unloaded Meso. Scale bar = 75 μm. (C): Quantitative analysis of neurite length from MNs on PDL/laminin-coated coverslips after direct administration of CNTF and GDNF, treatment with MesoMim, or treatment with unloaded Meso. Results from 7–10 experiments are expressed as mean ± SEM, and the MesoMim group is set at 100%. Direct and MesoMim administration of the factors induced a significantly greater extent of neurite outgrowth compared with the unloaded Meso group; ***, p < .001. No statistically significant differences were observed between groups with direct or MesoMim administration of the factors (p > .05). (D): HB9-GFP+ MNs expressed the MN markers ChAT and Isl1 in a 3-day differentiation assay after treatment with CNTF and GDNF or MesoMim but not in the absence of factors. Scale bar = 25 μm. (E, F): Almost all GFP+ cells expressed Isl1 (E) and ChAT (F) after treatment with CNTF and GDNF or MesoMim. Abbreviations: bTUB, β-tubulin; ChAT, choline acetyltransferase; CNTF, ciliary neurotrophic factor; D, day; GDNF, glial cell line-derived neurotrophic factor; GFP, green fluorescent protein; Isl1, Islet 1; Meso, mesoporous silica; MesoMim, mesoporous silica loaded with peptide mimetics; Rel., relative.

This potentially versatile and widely applicable strategy for the efficient differentiation and functional integration of stem cell derivatives upon transplantation, and it can serve as a foundation for improving stem cell-based neurodegenerative protocols, for example, Parkinson’s disease.

Alfonso Garcia-Bennett of Stockholm University, one of the lead authors of this study, said: “We are working to provide standard and reproducible methods for the differentiation and implementation of stem cell therapies using this type of approach, which coupled material science with regenerative medicine.”

Garcia-Bennett continued: “We demonstrated that delivering key molecules for the differentiation of stem cells in vivo with these particles enabled not only robust functional differentiation of motor neurons from transplanted embryonic stem cells but also improved their long-term survival.”

This research group is already working together with two companies to speed up the commercialization of a standard differentiation kit that will allow other scientists and clinicians to reproduce their work in their own laboratories.