Battens update 10/2017

We have shown that CdSe/ZnS Quantum dots solubilized with novel positively charged compact ligands (CL4) are not toxic to cells or mice. Others have replaced the 6nm QDs with gold nanoparticles (100nm) but these lack the trackable highly fluorescent core of the QD and tend to get stuck in the endosomal compartment. No one else has the CL4 QDs or is using our approach to delivering His-tagged recombinant proteins to cells and tissues from mouse models of lysosomal storage diseases (LSDs). As with most LSDs the perceived problem in Late infantile Batten disease (CLN2) has always been to get the enzyme (eg tripeptidylpeptidase TPP1) across the blood brain barrier (bbb) but a recent human clinical trial performed on 30 children has shown the direct injection of enzyme intraventricularly was able to stop disease progression. Although there has not been much recovery of lost functions the results are very encouraging and suggest either that this enzyme can cross the bbb or that the bbb is impaired in Battens CLN2 disease. We currently co-attach a cell-penetrating peptide (JB577) to the QD to overcome this problem and we have shown that JB577 also promotes endosomal egress - so that enzyme is delivered to the lysosome. The field of neural LSDs is currently focused on early diagnosis and a blood spot test has been shown to permit diagnosis of CLN2 kids days after birth—but there are 4 million births in the US each year.  Other labs are trying to repeat the BioMarin results in the other 60-100 lysosomal storage diseases now that the bbb may not be an issue.

Our current focus is on improving TPP1 delivery stability so that we do not need to treat with such a huge bolus of enzyme (300mg) every 2 weeks, ($500,000+/year). By targeting the TPP1 enzyme to neurons rather than astrocytes and microglia by means of a negatively charged coating (CL4) we can greatly improve the delivery of enzyme to brain. The protease TPP1 is an ancient enzyme and is found all the way back to amoebae and slime molds. The human version of TPP1 has developed complex regulatory mechanisms over the course of evolution, rather like the 1988 tax reform bill, so there is a chance to find drugs which will stimulate residual enzyme and reverse the inability to digest protein in the cells garbage recycling system -the lysosome. We care searching for clues by this approach by performing whole brain analyses of gene expression which should highlight any compromised pathway caused by protein indigestion in late infantile Battens disease.

            We are also continuing to work on the nature of the storage material in Batten disease since loss of stored material is the best biochemical measure of how well the therapy is working. We have shown that three oncofetal antigens GM3, GM2 and GD3 build up in CLN2 Battens brains-both humans and mice. These three show up in many LSDs and are hard to therapeutically remove. I worked on this problem for 6 months while on sabbatical in Adelaide, Australia 2006-2007 with John Hopwood’s group so I am eager to apply this knowledge to the mice which lack the protease TPP1.


Whats new with Sphingomyelinases?

Neutral sphingomyelinase2 (NSMase2) is active in brain and is induced by various types of stress. It is also believed to induce the formation of exosomes, the extracellular vesicles which contain bioactive RNA. The use of RNA interference (RNAi), which consists of miRNA (microRNA) and siRNA (small interfering RNA), is a potential approach to treat brain disorders by suppression of abnormal protein synthesis.  However, despite the inherent potential of these small nucleic acid based drugs, there is currently no efficient or safe system for their in vivo delivery. To address this problem, we report the successfully delivery of siRNA to inhibit sphingomyelinase synthesis. We used luminescent 6nm CdSe/ZnS Quantum Dots (QDs) which coated with zwitterionic or polyethylene glycol–based ligands and linked to the JB577 peptide (WG (Dap [Palmitoyl] VKIKK P9 GG His6).  We used a gel-shift assay to show that siRNA for luciferase and both the smpd1 (acid  (lysosomal) sphingomyelinase, ASMase), and smpd3 genes (NSMase2), are able to bind to JB577/QDs by electrostatic interaction through the three positively charged lysine residues (VKIKK) and negative charged phosphate on siRNA. Delivery to cytosol in human oligodendroglioma cells which overexpressed NSMase2 (HOG-NSMase2), fibroblasts which expressed ASMase and HCT-116 cells expressing luciferase were demonstrated by reduced enzyme activity. Delivery of siRNA-QDs complex was demonstrated through confocal microscopy imaging.  Both QD and Cy3 labeled siRNA were visible in the cell cytosol after a 24-hour treatment. These data suggest that QD-siRNA constructs are a robust model of in vivo siRNA delivery and are promising vehicles for neuro-targeted RNAi.

(Getz et al., 2016).

Whats new in Enzyme replacement therapy?

State of the Art in Enzyme replacement therapy (ERT)

Because of the success in treating non-neuropathic type 1 Gaucher Disease with enzyme replacement therapy ERT) and substrate-reduction therapy (SRT), together with the financial rewards of providing such therapy, there are at least 30 pharma/biotech companies trying to do ERT for some of the 60-plus lysosomal storage diseases that have been shown to cause devastating brain disease in humans. Thus far the success rate in animal models has been much higher than in humans and in fact very little has been achieved that is likely to improve the life of children afflicted by LSDs. A likely reason for these disappointing clincal results could be the inability to deliver the gene or enzyme to brain cells due to the tight blood-brain barrier in humans. The most popular solution to this challenge is to make lots of the missing enzyme and deliver it by injection into the cerebral spinal fluid every 2 weeks. There have been claims that this slows disease progression in late infantile battens (CLN2). A second approach is gene therapy in which a single injection of a viral construct designed to take over brain cells and reprogram them to make the missing enzyme. This is currently not allowed by the FDA (except for very small trials such as the grays and CLN6) because of a high risk to the patient (a liver fibrosis patient (Geysinger) died from an immune reaction following gene injection). It is also difficult to deliver virus to all brain regions so multiple holes must be drilled in the skull to facilitate multiple injections. 

Both therapeutic strategies have major roadblocks that could be overcome by our approach of developing a platform to facilitate delivery to the brain and deliver the missing enzyme to the lysosome in the neurons where the storage material is accumulating. We have chosen late infantile batten disease (CLN2) to show the efficacy of such an approach since it is the most common form of batten disease (1400 births /year in the USA), is caused by a genetic deletion of a single enzyme (tripeptidylpeptidase-1 (TPP1) that has unique markers and pathology of disease progression to help assess efficacy.

Nanoparticles as delivery agents.

For the past 6 years we have worked closely with a group at Scripps Research in San Diego (cell-targeting peptides) and the US Naval Research Labs in Washington DC (nanoparticles, NP) to optimize the attachment of the missing enzyme to a highly fluorescent metallic nanoparticle. These nanoparticles (photoluminescent QDS for development and gold, AuNPs, for ultimate therapeutic use) are about the size of large serum proteins are biologically stable, and provide the source of color to allow us to video our devices in animals ??

These metallic nanoparticles (Cdse/ZnS Quantum Dots or gold) (QDs and AuNPs) must first be solubilized by a coating them with organic molecules and identical coatings can be used for QDs and AuNPs). We have discovered that by varying the charge and structure of these coatings, we can target the QDS to different brain cells. Thus for Parkinson’s Disease you would want to target only dopaminergic basal ganglia neurons, for multiple sclerosis you would want to target only myelin-producing cells, whereas for battens disease you would want to target all brain cells. The metal-surface of the NP is critical because it tightly bonds to the Amino Acid Histidine. By genetically including a histidine tag to our manufactured recombinantly-generated TPP1 enzyme we can attach it to the QD. We can also use this approach to attach our patented cell-targeting peptide and because of the large surface area around the QD we can attach up to 25 enzyme molecules to a single NP which itself is the size of a large protein molecule. The components self-assemble at room temperature so after 30 minutes we are ready to inject the Nanoparticle with its life-saving cargo.  We have shown that the NP cargo complexes can be taken up by brain slice preparations so that we can first correct the lack of enzyme and the resultant pathology in slices from CLN2 batten mice brains. More excitingly we can inject the QD-complex intravenously into a mouse, show that it gets into the brain capillary system and eventually deliver enzyme to neurons.  We believe that this is the current state of the art for LSD therapy and we are developing it to treat Batten disease.

Whats new in substrate reduction therapy?

Many neurodegenerative diseases are caused by either the absence of specific proteins or aggregation caused by mutant proteins/peptides but delivery of corrective treatment is a major problem. We illustrate a possible solution by showing that His6-green fluorescent protein (GFP) is not taken up by human cerebral endothelial cell cultures unless we bind it to a CdSe/ZnS Quantum dot (QD)  co-labelled with a specific cell penetrating peptide (JB577: WG[Dap]Palmitoyl] VKIKK P9 GG His6). This should work for any His6-tagged protein although some peptides/proteins may need the inclusion of a polyproline spacer as shown for JB577. We then showed that the positively charged VKIKK sequence in QD-JB577 could allow small interfering siRNAs to bind to JB577, be taken up by cells (verified by gel-shift assay) and suppress the synthesis of specific proteins. We used luminescent 6nm CdSe/ZnS Quantum Dots (QDs) coated with zwitterionic or polyethylene glycol–based ligands to bind to siRNA for luciferase to supress luciferase expression by 70% in HCT116 cells engineered to overexpress luciferase, comparable to commercial transfection systems, but with minimal toxicity. Some differences in efficacy were observed between differently charged solubilizing coatings. We further verified the method by delivering antisense to smpd1 (acid  sphingomyelinase (ASMase) or smpd3 (neutral sphingomyelinase (NSMase2)), and showing supression of overexpressed or endogenous activity of either ASMase or NSMase by 60-70% in human glioblastoma (HOG) or skin fibroblasts.  Both QD and Cy3 labeled siRNA were visible in the cell cytosol for at least 48h suggesting that QD-JB577 QDs could deliver or supress proteins/peptides in brain cells.