Treating Alzheimer’s Disease: An investigation into the use of Gene therapy to simulate

Christchurch mutation
Emmanuel Mekasha

Ms. Leila Chawkat

Mentor Program Period 6

Tong Li, PhD

John Hopkins University

20 February 20

Treating Alzheimer’s Disease: An investigation into the use of Gene therapy to simulate

Christchurch mutation

Abstract:

Alzheimer’s Disease (AD) is a neurological disorder caused by an array of factors

ranging from diet and exercise to the genetics of an individual. Upon manifestation of the

disease, a patient begins to experience memory loss and motor control loss as the disease begins

removing the axons present within the white matter of the brain. In recent years, Alzheimer’s

disease has proven to be difficult to treat; however, treatment aimed at the genetic factors of

Alzheimer's Disease has yielded promising results. As a result, the paper investigates the APOE

gene, more specifically the Christchurch APOE mutation, and its possible genetic application to

a wider audience of patients at significant risk of Alzheimer’s Disease. Instigation of the genetic

environment would be done through gene therapy involving CRISPR use.

Table of Contents:

Introduction - 2

Literature Review - 3

Data Collection Methods - 7

Results and Analysis - 10

Treating Alzheimer’s Disease: An investigation into the use of Gene therapy to simulate

Christchurch mutation
Conclusion - 13

References - 14

I. Introduction

Alzheimer’s disease is a late activating neurodegenerative malady correlated with an

array of external factors, including a poor diet with a lack of vitamins and triglycerides and a

poor mental and physical exercise regime, and genetic factors, including specific genes located

within certain chromosomes, such as the APOE gene within chromosome 19. Alzheimer’s

disease disrupts the action potentials within a body, leading to the disruption of neurotransmitters

which disrupts communication between the neurons. This causes the devastating symptoms of

memory loss and motor control loss. Treatment for Alzheimer’s disease has proved difficult due

to its neurological component; however, the relationship between genetic factors, specifically the

APOE gene, and Alzheimer’s disease has been especially prevalent. In more detail, a mutation

within the APOE gene, referred to as Christchurch APOE homozygosity, has been demonstrated

to delay the manifestation of Alzheimer’s Disease within patients. Through simulation of an

internal environment similar to that of Christchurch APOE homozygosity in other patients using

gene therapeutic methods, treatment can be developed to push back Alzheimer’s Disease

manifestation in patients without the APOE mutation.

II. Literature Review

Treating Alzheimer’s Disease: An investigation into the use of Gene therapy to simulate

Christchurch mutation
In order to describe the detriment of Alzheimer’s Disease, an overlook of action

potentials in the neuron must be made. The action potential begins in either free axons in white

matter or the axon of the neuron. Upon stimulation by an external stimuli, depolarization begins

within the axon to raise the voltage of the cell by opening sodium protein channels to intake

sodium ions. The channel remains open until the axon of the neuron is saturated with sodium and

a minimum voltage of 40mV is reached (Aurel & Arthur, 2016). After these criterias are met,

repolarization begins and the potassium protein channels are opened. Potassium rushes out of the

neuron through facilitated diffusion and greatly lowers the voltage of the axon such that its lower

than its resting potential (Caron, 2018). As a result of its low voltage, hyperpolarization begins,

the potassium protein channels close, and the potassium sodium channels open as to exchange a

disproportionate amount of sodium for potassium ions to slightly increase the voltage to resting

potential (Cuyvers & Sleegers, 2016). This chain of depolarization, repolarization, and

hyperpolarization occurs down the axon and causes the emission of neurotransmitters from the

dendrites of the free body axon or neuron. These neurotransmitters interact with the dendrites of

another neuron and go through the action potential once again to repeat the process (Landin-

Romero & Kumfor, 2016). The uninterrupted transfer of information from neuron to neuron

should occur when the body is at homeostatic balance. Neurological pathologies tend to

compromise this system by either blocking the movement of neurotransmitters or causing

atrophies in the axons that produce the neurotransmitters. Alzheimer’s Disease is a neurological

disease that does the latter.

Alzheimer’s Disease disrupts the neurological transfer of signals through

neurotransmitters by causing atrophy primarily within the white matter of the brain (Lee &

Mackinnon, 2017). The brain is composed of both gray matter, which is mostly composed of
Treating Alzheimer’s Disease: An investigation into the use of Gene therapy to simulate

Christchurch mutation
active neurons that are first stimulated when an external factor arises, and white matter, mostly

composed of separate axons which transfer the signals of the active neurons of gray matter to

other active neurons in other portions of gray matter (Arboleda-Velasquez & Lopero, 2019).

Atrophy within the white matter portion of the brain by Alzheimer's Disease disrupts the transfer

of information by causing the damaging of those vital axons that transfer information from one

part of gray matter to another. By resulting in the malfunction of axons, Alzheimer's Disease

prevents neurotransmitters, vacuoles that contain information that move from dendrite to

dendrite, from transferring their signal between neurons to their intended destination. This is

done by disrupting the action potentials, consisting of depolarization, repolarization, and

hyperpolarization, that occur on the axons (Barth & Tomaselli, 2019). Such disruption in the

homeostatic balance of the body leads to the symptoms of memory loss and mood alterations.

Conversing about its causes, Alzheimer's Disease is correlated with a collection of external and

genetic factors. External factors include bad dietary choices, such as the lack of vitamins,

minerals, carbohydrates, and lipids, and a lack of physical and mental exercise. As for genetic

factors, a major gene correlated with the manifestation of Alzheimer's Disease is the APOE gene

(Hovav & Kaminker, 2014).

The APOE gene is present within chromosome 19 of the human genome. Currently, there

are three main variants of APOE that hold significance to Alzheimer's Disease, APOE2, APOE3,

and APOE4. The APOE2 version is generally correlated with low chances of manifestation of

Alzheimer's Disease at a younger age or higher chances of manifestation of Alzheimer's Disease

at an older age (Yu & Tan, 2014). APOE4, however, is associated with high chances of

manifestation of Alzheimer's Disease at a younger age through the activation of the PSEN1

protein that leads to the buildup of amyloid beta plaques that cause atrophy within the white
Treating Alzheimer’s Disease: An investigation into the use of Gene therapy to simulate

Christchurch mutation
matter of the brain. APOE3, contrary to APOE2 and APOE4, has been correlated with little to no

change in the manifestation of Alzheimer's Disease (Weickenmeier & Jucker, 2018). The PSEN1

protein experiences no activity among APOE3. Interestingly, with APOE3, there exists a rare

mutation in this variant of the gene referred to as Christchurch APOE3 (APOEch). Patients with

two APOEch alleles seem to experience a delayed manifestation of Alzheimer's Disease. A

patient with a seemingly genetic Alzheimer's Disease due to the prevalence of Alzheimer's

Disease in the patient’s family did not contract Alzheimer's Disease until her seventies (Raj &

Yin, 2017). Such delayed manifestation is similar to that of a cure. Said patient had this irregular

mutation of APOEch homozygosity within her genome, leading to the conclusion of the

significance of the Christchurch APOE mutation in delaying Alzheimer’s Disease. Application

of this mutation in non carriers can be achieved through genetic alteration of the genome in order

to simulate an environment similar to one created by APOEch homozygosity within said patient.

Gene therapy may achieve this goal.

Gene therapy, the process in which gene editing occurs within an organism, would be the

optimal method of simulation of APOEch homozygosity within a patient (Pierzynowska &

Wegrzyn, 2018). For optimal insertion of the two APOEch alleles, a combination of the use of

CRISPR technology and a retrovirus would be best. Beginning with CRISPR use, CRISPR,

standing for clustered regularly interspaced short palindromic repeat, provides an RNA sequence

for which the Cas9 enzyme can accurately cut and insert genes (Gillespie & Jones, 2017). Said

RNA sequence would be deployed among the APOE gene, unzipping the double helix structure

to bind with one helix strand. Upon binding on the strand, the Cas9 enzyme can then go to work

by splicing the DNA in order to insert the two APOEch alleles into the genome. Paired alongside

the CRISPR technology would be the use of a retrovirus. The retrovirus bears similarities to the
Treating Alzheimer’s Disease: An investigation into the use of Gene therapy to simulate

Christchurch mutation
mechanism of viruses (Wisniewski & Drummond, 2020). Viruses, upon infiltration of the cell,

will direct itself to the nucleus or genetic material of the cell. The virus will then insert its own

genetic material in the double helix structure of DNA in the cell. The genetic material of the

virus will bind to the DNA, so that the genetic material of the virus can be replicated through

mitosis and activated through translation and transcription. Mirroring the mechanism of a virus,

the retrovirus acts as a vector for gene therapy and inserts genes or alleles into the genome. It

does so by being composed of three essential genes, gag, pol, and env, followed by terminal

repeats that allow for integration of manipulated viral genome for transcription and translation

into amino acids by the organism. A retrovirus containing two alleles of APOEch can be

introduced to the genome as to integrate APOEch naturally. A combination of the use of a

retrovirus and CRISPR to insert APOEch homozygosity within the genome hopes to simulate a

similar genetic environment as was present within the patient with APOEch homozygosity in an

Alzheimer abundant family. Through transcription and translation, simulated APOEch

homozygosity through gene therapy using CRISPR and retroviruses should prevent the

manifestation of atrophy created by amyloid plaques or the PSEN1 gene/protein in patients in

Alzheimer abundant families.

III. Data Collection Methods

Meta analysis was the optimal data collection strategy due to the resources and time

present. Ideally, an experimental data collection would be best as it allows for direct testing of

the thesis through an experiment with genetically altered mice with CRISPR instituted APOEch
Treating Alzheimer’s Disease: An investigation into the use of Gene therapy to simulate

Christchurch mutation
homozygosity and the production of results that would precisely prove or contradict the

argument. However, as stated previously, necessary resources include, but are not limited to,

CRISPR access, retrovirus access, a human genome with APOEch homozygosity, a multitude of

mice, and a laboratory, and these resources are not available as of currently. An observational

data collection strategy was not used for the same reason, a lack of resources. Data collection

involving surveys and interviews was not used as it possesses no practicality within an argument

attempting to support the implementation of gene therapy for Alzheimer’s Disease. Meta

analysis, instead, was used as it allowed for the collection of a multitude of studies to piece

together an argument establishing the significance of APOE within Alzheimer’s Disease, the

ability for APOEch to delay late onset Alzheimer’s Disease, and the practicality in using

CRISPR to insert the two alleles. Meta analysis allowed for the proving of the argument despite

the limited resources present.

Sources Jin-Tai Yu, Lan A. M. Saunders, Beatriz Candás‐ Yuxuan Wu and

Tan, and John W. J. Estébanez. Hai Zhou

Hardy. Strittmatter, D. Ariadna Padró‐ Correction of a

Apolipoprotein Schmechel, P. H. Miquel. APOE genetic disease

E in Alzheimer's St. George- Variants E2, E3, by CRISPR-

Disease: An Hyslop. and E4 Can Be Cas9-mediated

Update Association of Miscalled By gene editing in

apolipoprotein E Classical PCR‐ mouse

allele ϵ4 with RFLP When The spermatogonial

late‐onset Christchurch stem cells

familial and Variant Is Also

Treating Alzheimer’s Disease: An investigation into the use of Gene therapy to simulate

Christchurch mutation
sporadic Present

Alzheimer's

disease

Criteria Study examining Study examining Study examining Study examining

300 patients with families one patient and mice with the

Alzheimer’s seemingly comparing said implementation

Disease, and possessing patient’s heparin of

their genomes, sporadic binding and spermatogonial

specifically the Alzheimer’s LDR1 binding to stem cells

APOE genes. Disease and the other patients through genome

prevalence of and other APOE alteration

APOE4 within variants. through the use

said groups. of CRISPR

Data Research among Analysis of the Analysis of Troubles in

hundreds of genome of said genome and activation and

patients families has brain tissue of the prevention of

possessing discovered that patient reveals mutation using

Alzheimer’s clusters of APOEch alternate

Disease reveals sporadic homozygosity methods have

that a majority Alzheimer’s has reduced proved

developed late Disease seems to LDLR binding prevalent.

onset be correlated by Mutation of the

Alzheimer’s with the approximately EGFP transgene

Treating Alzheimer’s Disease: An investigation into the use of Gene therapy to simulate

Christchurch mutation
Disease (LOAD) possession of the 60%, using CRISPR

and APOE4 gene, comparable to technology to

approximately with 36%-40% the reduction an highlight

80% of the of sporadic active APOE2 specific portions

Alzheimer’s Alzheimer’s gene possesses at of the DNA to be

Disease patients Disease patients 90%, and has altered by the

seemed to have possessing the reduced heparin Cas9 enzyme

genetics and gene. binding, a factor have, however,

heritability play with influence in proved

a key role in its the manifestation successful in

manifestation. of amyloid beta altering the

Among the plaques, genome of used

genetic factors of considerably. mice.

Alzheimer’s Although the

Disease, patient's brain

Apolipoprotein samples

E seems to be demonstrated

among the most high amyloid

prevalent. plaque burden,

the patient did

not experience

atrophy.
Treating Alzheimer’s Disease: An investigation into the use of Gene therapy to simulate

Christchurch mutation
IV. Results and Analysis

Study 1’s results, among 300 patients, conclude with the idea that most Alzheimer’s

Disease patients develop LOAD, late onset Alzheimer’s Disease. With this revelation, it was

determined that 80% of Alzheimer’s Disease patients seem to have their genetics heavily

influence the manifestation of late onset Alzheimer’s Disease. As the APOE gene has been

identified as a crucial genetic factor, it stands to reason that therefore, the APOE gene possesses

large effects on the manifestation of late onset Alzheimer’s Disease. This statement is supported

by Study 2’s results, among members of families with seemingly sporadic Alzheimer’s Disease,

which identify APOE4 as between 36% to 40% common among the patients with sporadic

Alzheimer’s Disease. This once again reinforces the idea that the APOE gene heavily impacts

the manifestation of Alzheimer’s Disease. Both study 1 and study 2 aim to demonstrate the

significance of the APOE gene, specifically APOE4, in Alzheimer’s Disease, and therefore, the

ability of a genetic treatment to delay the manifestation of Alzheimer’s Disease. This can be

provided through the proposed treatment of gene therapy to institute APOE3ch homozygosity.

Study 3’s results, among a patient with two Christchurch APOE3 alleles in an Alzheimer

abundant family, demonstrate decreased heparin binding and LDLR binding by 60%, signifying

low atrophy in comparison to Alzheimer’s disease patients. Although the patient seemed to not

be relieved of amyloid beta plaque burden, atrophy was not present within the brain. This serves

to demonstrate the ability of APOEch homozygosity in constructing an environment with a lack

of atrophy, and therefore, its ability as a treatment that may be implemented through gene

therapy. Finally, study 4’s results, among mice with the implementation of spermatogonial sperm

cells, demonstrates the efficiency of the use of CRISPR technology to highlight and edit genes

for removal or implementation. Success in the implementation of spermatogonial sperm cells

Treating Alzheimer’s Disease: An investigation into the use of Gene therapy to simulate

Christchurch mutation
through CRISPR technology provides promise in the use of it for insertion of APOEch

homozygosity. Together, all four studies construct an argument in which the basis for genetic

alteration is created, as a result of the significance of genetic factors, specifically APOE, within

Alzheimer’s Disease as established by study 1 and study 2, the reasoning for implementation of

APOEch homozygosity is constructed, as a result of its trait of delaying the manifestation of

Alzheimer’s Disease within an Alzheimer’s Disease prevalent family as indicated by a patient

present in study 3, and the ability for CRISPR to institute the change is established, as it proved

vital in instituting a change in the genome for spermatogonial stem cells within multiple patients

in study 4.

Limitations are present in the Data Collection, mostly stemming from the chosen meta

analysis strategy. The four chosen studies are not meant to be looked at together, and therefore,

there exists small inaccuracies in the conclusions developed from the results of said studies. The

inaccuracies have been minimized by comparing studies with similarities present within the

structure, such as with the examination of the APOE gene within study 2 and study 3. However,

if this was to be done again, studies with more similarities in structure would be assembled for

analysis.

Together, all four studies reinforce the argument about implementation of APOEch

homozygosity through gene therapy using CRISPR by proving specific portions of the claim.
Treating Alzheimer’s Disease: An investigation into the use of Gene therapy to simulate

Christchurch mutation

V. Conclusion

Alzheimer’s disease severs the modes of transportation of the body’s neurotransmitters

by creating atrophy constructed by amyloid beta plaques within the free axons in the white

matter of the brain. This causes the loss of homeostatic encounters in the neurological state of a

patient and results in effects such as memory loss, behavioral changes, and motor loss control.

Promising results, however, have been demonstrated through Christchurch APOE homozygosity

in several patients in Alzheimer abundant families as they demonstrated a lack of manifestation

of Alzheimer’s Disease. Thus, implementation of APOEch homozygosity would be ideal and

would prove best through CRISPR use, a method proved successful through spermatogonial

sperm cell experiments, and retroviruses, which institute DNA biologically. Through the

implementation of APOEch homozygosity through these methods, the manifestation of

Alzheimer’s Disease can be delayed, and Alzheimer’s Disease can be treated..

Treating Alzheimer’s Disease: An investigation into the use of Gene therapy to simulate

Christchurch mutation

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