Possible New Therapies for Deadly Neural Tube Defects!

Neural Tube Defects (NTDs) are a type of birth defect that can impact the brain, spine, or spinal cord during the early stages of fetal development (1). Common NTDs that fetuses develop include Spina Bifida, Anencephaly, Encephalocele, and Iniencephaly (5,6). NTDs have a strong global prevalence, impacting approximately 322,000 pregnancies worldwide annually (2). In the United States alone, NTDs occur in approximately 3,000 babies annually, with 1,000 of those affected experiencing Anencephaly (1,3,4). NTDs are particularly challenging to treat early, with no effective treatments, as the defects typically begin developing during the first month of pregnancy, which is when some women may not even know they are pregnant (1). Unfortunately, by the time Neural Tube Defects are correctly and definitively diagnosed, it becomes extremely difficult to effectively treat it. Prior to researching more, I had never heard of Neural Tube Defects! However, being a Biomedical Engineer, I only felt that it was my role and responsibility to contribute at least a little bit of the principles that I have learned to come up with new therapies! Who knows? Maybe, it can help someone one day. So, in this article, I decided to highlight what genes I thought of targeting so far. While this may turn out to be a nothing burger, hopefully it’s at least an interesting read for you all!

Previous literature that I read through established the FOX1, ANKDR6, and WDR4 genes as significant contributors to the development of neural tube defects. Based on this, I came up with two methods by which normal functioning of these genes can be restored, and NTD development can be prevented, both carried out before the 6th week of the first trimester of pregnancy, since that is when the neural tube closes. Each of the genes being targeted plays a significant role in normal transcription regulation/signaling mechanisms that, when disrupted, lead to NTD’s. ANKDR6 mutations affect normal reciprocal mechanisms, causing motor neuron pools to be deactivated, hindering muscle innervation (7). WDR34 mutations affect the PCP signaling pathway, impacting muscle regeneration and neuron orientation (9,10,11); 3). FOX1 gene mutations lead to the formation of abnormal FOX1 proteins, which affects downstream transcriptional regulation of NTF mechanisms (8,12). The rationale for having two methods is that a comparison of both the methods will provide insight into which one is more effective in preventing NTDs over the long-term, especially given the known limitations of synthetic oligonucleotides for gene editing and removal. These aims will be tested on pregnant mice, whose fetuses have mutations in these three genes. 

As you can see below, based on the research that I did, I compiled a few more details for each of my aims. These are works in progress, but using my creative mind, this is what I was able to think of!

Aim 1: Utilize synthetic oligonucleotides with Homology Directed Repair to edit genetic mutations in the FOX1, ANKDR6, and WDR4 genes to prevent the formation of Neural Tube Defects.

1. Mice will be randomly assigned to treatment, placebo, and control groups. 
2. For the treatment group, complementary DNA Oligonucleotides to the genes will be created, while those unrelated to the three genes will be created for the control group and none will be created for the placebo group (13).
3. Synthesize Repair DNA template strands, with Fluorescent Reporter Genes (GFP, BFP, MCherry), using promoters. They will then be ligated/joined with the oligonucleotides (14,20,21).
4. The oligonucleotides with the fluorescent reporter genes will be successfully delivered to the somatic cells of the fetuses, through lipid nanoparticles (18,19). This will facilitate a double strand break at the ends of each of the three genes, which will promote their deletion, due to the HDR Mechanisms present in the cell (15,16,17).
5. After enough time has passed, these cells will be harvested, and inserted through a flow cytometer. MFI will be measured, with a higher MFI, correlating to a higher success of gene removal (23).

Aim 2: Utilize Crispr-Cas9, with HDR via a Lipid Nanoparticle delivery system to edit genetic mutations in the FOX1, ANKDR6, and WDR4 genes to prevent the formation of Neural Tube Defects.

1. Mice will be randomly assigned to treatment and control groups.
2. For the treatment group, guide RNA strands that correspond to the three genes will be created, while nonspecific guide RNA strands will be created for the control group (16).
3. Synthesize Repair DNA template strands, with GFP, BFP, MCherry, using promoters (14). The Cas9 proteins, the gRNA and the repair template DNA will be delivered, through lipid nanoparticles (18), to the somatic cells of the fetuses. This will facilitate the breakage of the strands containing these genes, and their substitution with strands that lack these genes and express proteins that induce fluorescence (15,16,17). 
4. After enough time has passed, these cells will be harvested, and inserted through a flow cytometer. Mean fluorescence intensity will be measured, with a higher MFI, correlating to a higher success of gene removal.

My hope is that my approach of editing mutated versions of these genes in fetus somatic cells and replacing them, can inspire some scientists to pursue this further, and help to prevent NTD development and save lives. The problem is that, right now at least, if the genes were not edited or replaced, children would inevitably not survive the pregnancy, due to the lack of any effective treatments. These types of diseases simply do not have effective cures, and they require out of the box thinking to make any progress. Conventional wisdom does not always work in science! There are limitations, and challenges down the road, however. Edits will not automatically be passed on to future generations, since they do not affect the germline cells, which means that the offspring of someone who has this gene in their genome will unfortunately still suffer the consequences (19). This is a problem that I unfortunately could not find a way to get around in my methods, but hopefully I have provided a good starting point for other researchers to look into! If you made it this far, kudos to you and hopefully this inspires you to look into these horrible diseases as well!

Sources:

1. Cleveland Clinic Medical Professional. Neural tube defects (NTDs): What They Are, Causes & Prevention. Cleveland Clinic. March 30, 2022. Accessed October 28, 2023. https://my.clevelandclinic.org/health/diseases/22656-neural-tube-defects-ntd. 
2. Kancherla V. Neural tube defects: a review of global prevalence, causes, and primary prevention. Child’s Nervous System. 2023/07/01 2023;39(7):1703-1710. doi:10.1007/s00381-023-05910-7
3. Neural Tube Defects. March of Dimes. February 2022. Accessed October 28, 2023. https://www.marchofdimes.org/find-support/topics/planning-baby/neural-tube-defects#:~:text=Screening%20tests%20for%20NTDs%20include,to%2022%20weeks%20of%20pregnancy. 
4. MedlinePlus [Internet]. Bethesda (MD): National Library of Medicine (US); [updated 2020 Jun 24]. Anencephaly; [updated 2019 Oct 01; reviewed 2018 Jun 01; cited 2023 Oct 29]; [about 5 p.]. Available from: https://medlineplus.gov/genetics/condition/anencephaly/. 
5. King, Jesse, “Anencephaly”. Embryo Project Encyclopedia ( 2013-02-13 ). ISSN: 1940-5030 https://hdl.handle.net/10776/4215
6. Amorosi S, D’Armiento M, Calcagno G, et al. FOXN1 homozygous mutation associated with anencephaly and severe neural tube defect in human athymic Nude/SCID fetus. Clinical Genetics. 2008;73(4):380-384. doi:https://doi.org/10.1111/j.1399-0004.2008.00977.x
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