This post explores three different strategies to correct defects in two members of the CLCN family of H+/Cl– lysosomal anti porters.
Antisense oligonucleotides 
Most patients with CLN3 Batten have a deletion encompassing exons 7 and 8 (CLN3Δex7/8), creating a reading frame-shift . The deletion of exons 7 and 8results in a premature termination codon in exon 9 of the 15-exon gene. The authors from the Rosalin Franklin University of Medicine were challenged with correcting for a mutation that removed two large protein coding regions as well as a premature termination codon on exon 9. UGA and UAA are codons that tell the ribosome to stop translating the mRNA into protein.
Exon 5-targeted ASO-induced robust exon skipping for more than a year, improved motor coordination, reduced histopathology in Cln3Δex7/8 mice and increased survival in a new mouse model of the disease. ASOs also induced exon skipping in cell lines derived from patients with CLN3 Batten disease. Our findings demonstrate the utility of ASO-based reading-frame correction as an approach to treat CLN3 Batten disease and broaden the therapeutic landscape for ASOs in the treatment of other diseases using a similar strategy.
Adenovirus delivery 
A group from the University of Nebraska used self-complementary adeno-associated virus 9 (scAAV9) directed gene therapy to treat a mouse model of Batten Disease. Unlike regular single stranded DNA adeno associated virus, this one is double stranded.
A promoter is a region of a piece of chromosomal DNA upstream of the part of the gene that codes for the protein. Proteins called transcription factors bind to the promoter such as to facilitate the binding of RNA polymrease that copies the nucleotide sequence of the protein coding part of the gene into messenger RNA. mRNA is later translated into protei by the ribosomes.
- methyl-CpG-binding protein 2 (MeCP2) is a protein that is expressed in low amounts in most? tissues 8x increase in CLCN3 expression
- β-actin is expressed in large amounts in just about every tissue and is claimed to have a strong promoter. 3x increase in brain.
This approach was based on the expectation that low CLN3 levels are required for cellular homeostasis due to minimal CLN3 expression postnatally, although this had not yet been demonstrated in vivo.
These mice had had two protein coding segments, exons, seven and 8 removed from the CLCN7 gene. Δex7/8. These mice had similar disease characteristics as human patients: disease phenotypes, including motor deficits, glial activation, and progressive accumulation of lysosomal storage material.
One-month-old Cln3Δex7/8 mice received one systemic (intravenous) injection of scAAV9/MeCP2-hCLN3 or scAAV9/β-actin-hCLN3, with green fluorescent protein (GFP)-expressing viruses as controls.
Only the scAAV9 construct driving low CLN3 expression (scAAV9/MeCP2-hCLN3) corrected motor deficits and attenuated microglial and astrocyte activation and lysosomal pathology.
This may have resulted from preferential promoter usage because transgene expression after intravenous scAAV9/MeCP2-GFP injection was primarily detected in NeuN+ neurons, whereas scAAV9/β-actin-GFP drove transgene expression in GFAP+ astrocytes. This is the first demonstration of a systemic delivery route to restore CLN3 in vivo using scAAV9 and highlights the importance of promoter selection for disease modification in juvenile animals.
Silencing RNA, siRNA 
Capuli and coworkers of Italy chose to look at several missense mutations in the CLCN7 genes.
Disease causing point mutations are shown.
The goal was to design complementary stretches of siRNA that will bind with high affinity to the mutant sequence but not to the wild type sequences in a cell culture system.
The jetPEI® transfection reagent, a linear polyethylenimine derivative, free of components of animal origin, was used for gene delivery. The stability in mice was also determined.
treatment of ADO2 osteoclasts with Clcn7G213R-siRNA reduced the transcriptional expression of Clcn7G213R, without affecting the Clcn7WT transcript nor the expression of other genes of the Clcn family, such as Clcn5 and Clcn5
Clcn7G213R/WT mice were treated with the siRNA PEI conjugates three times a week for 2 and 4 weeks. No pathological issues were noted upon necroscopy. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed that Clcn7G213R mRNA expression was reduced in all organs tested but the brain. The authors interpreted this as no crossing of the blood–brain barrier.
Note that while the brain is not targeted by the siRNA, other organs are. Clalvaria bone mRNA was silenced by about 50% while femur bone mutant CLCN7 transcripts were silenced by much more. Capulli and coauthors some dramatic improvements in fumur health.
Capulli and co-authors grew human breast cancer MDA-MB-231 cells transfected with the CLCN7R767W vector as subcutaneous tumors in athymic mice. (These mice cannot produce antibodies.) The breast cancer tumors were sensitive to CLCN7R767W-specific siRNA treatment in vitro, showing a specific concentration-dependent down regulation of ClCN7R767W-EGFP chimeric mRNA. Mice where then treated with a single i.p. injection of CLCN7R767W-specific siRNA. The tumors were removed after 96 hours and examined for CLCN7R767W-EGPF chimeric RNA expression by RT-PCR. Less CLCN7R767W-EGPF mRNA in tumors was observed in response to CLCN7R767W-specific siRNA treatment, Scrambled siRNA had no effect.
The exciting questions would be the impact of this CLCN7R767W mutation on breast cancer cell autphagy. Is it increased or decreased? Did the siRNA reverse the affect?
Two entirely different types of mutation, three strategies
The large scale CLCN3 deletion mutations of Batten’s Disease require different strategies as this deletion of the C-terminus half results in a dysfunctional protein. One strategy is to introduce a functional version with an adenovirus.  The other is to use anti-sense RNA to remove part of the central part of the protein while re-acquiring the C-terminus.  Gain of function CLCN7 mutations can be silenced with siRNA  The only challenge is to get the siRNA past the blood brain barrier. 
- ACenta JL, Jodelka FM, Hinrich AJ, Johnson TB, Ochaba J, Jackson M, Duelli DM, Weimer JM, Rigo F, Hastings ML. Therapeutic efficacy of antisense oligonucleotides in mouse models of CLN3 Batten disease. Nat Med. 2020 Sep;26(9):1444-1451 PMC free article
- Bosch ME, Aldrich A, Fallet R, Odvody J, Burkovetskaya M, Schuberth K, Fitzgerald JA, Foust KD, Kielian T. Self-Complementary AAV9 Gene Delivery Partially Corrects Pathology Associated with Juvenile Neuronal Ceroid Lipofuscinosis (CLN3). J Neurosci. 2016 Sep 14;36(37):9669-82. PMC free article
- Capulli M, Maurizi A, Ventura L, Rucci N, Teti A. Effective Small Interfering RNA Therapy to Treat CLCN7-dependent Autosomal Dominant Osteopetrosis Type 2. Mol Ther Nucleic Acids. 2015 Sep 1;4(9):e248. PMC free article