Precision Genome Editing towards the Treatment of

Hemoglobinopathies

Huimeng Sun

a

Queen Margaret’s School, Duncan, British Columbia, Canada

Keywords: CRISPR-Cas9, Base Editing, Sickle Cell Anemia, β – Thalassemia, Delivery Methods, Clinical Trials.

Abstract: Hemoglobinopathies, including sickle cell disease and β – thalassemia, are genetic disorders that cause

people to suffer from anemia. Apart from the lifelong therapeutic methods, gene therapy has been

introduced in the last decades of research as an efficacious treatment option, supported by various types of

delivery methods. In this work, I review the precision genome editing towards the treatment of

hemoglobinopathies. With a brief cover of the disease pathology and genome editing tools, special focus has

been directed towards the potential editing sites and clinical trials in progress.

1 INTRODUCTION

1

According to the World Health Organization

(WHO), approximately 7% of the global population

are carriers of hemoglobinopathies, which are

divided into thalassemia syndromes and structural

hemoglobin variants. The most common

hemoglobinopathies are β-thalassemia and sickle

cell disease (SCD). Patients suffering from β-

thalassemia and sickle cell disease (SCD) show

mutated or low levels of β-globin chain production.

The mutated β-globin chains result in the

hemoglobin tetramer to polymerize, which causes

the red blood cells to create a “sickle-like” shape

leading to vaso-occlusive crises and tissue damages.

Allogeneic hematopoietic stem cell (HSC)

transplantation together with iron chelation is the

only approved curative treatment for severe genetic

blood disorders. However, this method is

constrained by the availability of human leukocyte

antigen matching donors and face the limitation of

graft rejection. Collectively, these genetic blood

disorders are recognized as one of the major health

challenges in the world. which results in low

flexibility and induces hemolytic anemia and

vascular occlusions.

Over the past decade, new technological

breakthroughs in genome editing have propelled the

potential to cure genetic disorders. A cutting-edge

a

https://orcid.org/0000-0001-8322-2845

technology adapted from the CRISPR (clustered

regularly interspaced short palindromic repeats)/Cas

(CRISPR-associated protein) bacterial immune

system has been established as a platform for

performing more efficient, accurate, and precise

genome editing. These fundamental technologies

have led to the creation of groundbreaking tools that

have the capability to transform biological research

and curing genetic diseases. Traditional CRISPR

editing employs the creation of double stranded

breaks (DSBs) by endonucleases, which

subsequently stimulates DNA repair mechanisms.

These numerous developments have accelerated the

exploration of various curative strategies for the

treatment of hemoglobinopathies. Genome editing

approaches that generate DNA DSBs can correct the

disease-causing mutation or induce higher levels of

fetal hemoglobin (HbF) expression. However,

nuclease-mediated DSB-induced cytotoxicity and

chromosomal rearrangement severly limit these

approaches as eventual treatments. Recently

developed DSB-free strategies have overcome these

issues while maintaining superior clinical outcomes.

There are now many parallel clinical trials that

explore multiple editing strategies for the treatment

of β–hemoglobinopathies. Clinical studies using

lentiviral-based gene modifications have proved to

be successful at ameliorating the clinical symptoms

in patients with hemoglobinopathies. The

transplantation of genetically modified autologous

hematopoietic stem cells (HSCs) prevents any

immunological risks, such as graft reject. The

Sun, H.

Precision Genome Editing towards the Treatment of Hemoglobinopathies.

DOI: 10.5220/0011377400003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 1065-1075

ISBN: 978-989-758-595-1

Copyright

c

1065

majority of the patients receiving edited HSCs

demonstrate reduced transfusion requirements while

maintaining high levels of fetal hemoglobin

expression. In this review, I present a comprehensive

overview comparing different types of genome

editing technologies and approaches for the

treatment of β–hemoglobinopathies.

2 OVERVIEW OF DISEASE

PATHOLOGY (SICKLE CELL,

BETA-THALASSEMIA, ETC.)

Sickle Cell Disease (SCD) is an autosomal recessive

genetic disorder affecting the function of

hemoglobin and was first discovered in 1910 by

James Bryan Herrick. Hemoglobin is tetrameric

molecule that formed by a globin group surrounded

by four heme groups and function as carriers of

oxygen (O2), carbon dioxide (CO2), and nitric oxide

(NO). They are critical in transferring oxygen (O2)

throughout the bloodstream from lungs to tissues

and cells, which is a major distinction of mammal

life. Sickle cell anemia is caused by an inherited,

single missense mutation in the β globin chain. The

single adenine to thymine base substitution results in

an amino acid conversion from glutamine to valine

in the globin gene. Normal red blood cells are

shaped like a binocave discoid, providing elasticity

to the cell and allowing red blood cells to pass

through the capillary bed to deliver oxygen.

However, the deoxygenation of sickle hemoglobin

(Hb S) results in an abnormally shaped erythrocyte

with a “sickling” appearance. This results in a

polymerization of sickle red blood cell which results

in low flexibility and induces hemolytic anemia and

vascular occlusions.

The β–thalassemia and α–thalassemia are two

major categories of thalassemia syndromes, in which

the patient lacks the production of β-globin chain. α–

thalassemias are a result of defects in the α–globin

chain of adult hemoglobin, including partial (α

+

) and

complete (α

0

) deletions or mutations of the four α–

globin genes. α –thalassemia can be further

categorized into 4 types based on different

genotypes and symptoms. α thalassemia silent

carriers are when only one gene is deleted or

damaged out of three; because only a single copy is

affected, the carrier does not show any clinical

symptoms nor a reduction in Hb values, but the

affected gene is inheritable through generations. α

thalassemina carriers have two missing genes that

result in mild anemia. Hemoglobin H disease is

caused by three missing genes that may lead to

moderate and severe microcytic hydrochronic

anemia resulting in impaired hemoglobin

production, with symptoms of fatigue, exercise

intolerance, and enlarged spleens. α - thalassemia

major is a fatal disease without treatment, in which

all four genes are missing that cause severe anemia.

β - thalassemia are caused by the insufficient (β

+

) or

absent (β

0

) production of β-globin chains in

hemoglobin and is highly prevalent in

Mediterranean countries. β- thalassemia can be

further divided into 3 types. Carriers of β–

thalassemia minor are normally clinically

asymptomatic. β- thalassemia intermediate cases

cause milder anemia and the majority of patients do

not require any blood transfusions to reduce chronic

anemia due to a compensation mechanism of

hypertrophy by erythroid marrow. β- thalassemia

major corresponds to symptoms of growth

retardation, progressive enlargement of abdomen,

and skeletal deformities. Although regular

transfusion medications are available, they may

result in problems of iron overloading which cause

dilated myocardiopathy, fibrosis, diabetes, etc.

Cardiac diseases are the most life-threatening

complication in patients treated with blood

transfusions.

3 OVERVIEW OF GENOME

EDITING (ZNF, TALEN, CAS9,

BASE EDITING, PRIME

EDITING, AND MORE)

Over the decades, the emergence of gene editing

technologies has offered scientists the ability to

introduce modifications to the genome of various

cell types in hopes of creating genetic therapeutics.

The use of highly specific and programmable

nucleases that introduce double-stranded breaks

(DSBs) provides the ability to specifically edit any

the region of interest. DSBs are repaired by

endogenous cellular mechanisms, either through a

non-homologous end-joining (NHEJ) repair pathway

that generates insertion and deletions (indel), or

homology-directed repair (HDR) pathway that

utilizes a genetic donor template to replace the DNA

surrounding the DSB. Meganucleases (homing

endonucleases), the earliest gene technology, are

sequence-specific DNA cleavage enzymes used in

targeting, replacing, and modifying the genome.

Meganucleases, exist as dimers or single-chain

enzymes, bind and cleave sequences that are at least

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1066

12 base pairs in length with high specificities.

Although effective at manipulating DNA,

meganucleases have one major limitation. The

biggest limitation is that for a meganucleases to bind

to DNA, the protein requires specific sequences

upon which only this protein can bind to the

matched DNA sequence. If one wishes to change the

target DNA sequence, a whole different protein, or a

profound protein engineering effort of the

meganucleases is needed to evolve and re-engineer

the protein, which makes it difficult for widespread

usage of these tools across biomedical research and

biomedicine.

The next two big classes of gene editing

technologies developed are zinc finger nucleases

(ZFN) and transcription activator-like effector

nucleases (TALEN). ZFNs consist of engineered

zinc fingers fused to a non-specific, dimeric FokI

nuclease domain. DNA-contacting residues can be

replaced by connecting individual zinc fingers with

novel DNA-binding specificities. However, there is

a grand challenge when making multi-finger arrays

as the fingers are not always modular. Each

individual zinc finger recognizes a specific 3-base

pair sequence, but each finger also has context-

dependent effects, so it is not always as simple as

replacing the finger with the desired targeting

sequence. TALENs are modular DNA-binding

protein units derived from naturally occurring

TALEs (from Xanthomonas). Each monomeric

TALE repeat recognizes a specific type of DNA

base. Like zinc finger arrays, individual TALE

subunits can be simply joined together to recognize

longer sequences of DNA. Similar to ZFNs, TALE

repeat arrays can be fused to a dimeric FokI nuclease

domain to create TALENs. Several differences are

noted between ZFNs and TALENs. The biggest

distinction between them is that each monomeric

TALE subunit binds to only a single nucleotide

instead of a nucleotide triplet, which makes TALE

targeting simpler than that of ZFNs. From a cost and

time perspective, TALENs are cheaper and faster to

produce, and are more flexible and easier to design

because of their simple DNA recognition properties

compared to ZFNs. Immunogenicity is another huge

consideration for using ZFNs and TALENs in

therapeutic applications. ZFNs display little to no

immunogenicity because the sequences are found in

all organisms from yeast to humans, while the

immunogenicity for TALENs in humans is still

unknown. However, it is predicted that the immune

response of TALENs may be relatively higher since

the sequences of TALE repeats are only found in

Xanthomonas plant pathogens.

Although meganucleases, ZFNs, and TALENs

all have their distinct advantages, there are still

several limitations remaining to their widespread.

The primary difficulty in using these technologies is

that they are fundamentally dictated by a protein-to-

DNA interaction when driving specificity of DNA

targeting. Therefore, an extensive protein

engineering and optimization effort is needed when

one wants to target a new sequence of DNA.

Furthermore, the cost and time needed to produce

each variant is a huge limitation that needs to be

considered. It takes weeks to months to produce one

specific agent that then still needs to be optimized

for a specific site, and many times prices go beyond

the financial capability of people and researchers

with goals of routine usage. Therefore, there remains

a need for an alternative approach that can overcome

the remaining limitations in genome editing.

Clustered regularly interspaced short

palindromic repeats (CRISPR) and CRISPR-

associated protein 9 (Cas9) is the latest technology

that revolutionized the field of genome editing.

CRISPR-Cas9 was first identified in 2008 as a

bacterial defense mechanism that host bacteria

leverage against bacteriophage infections in nature.

This was later repurposed as a gene editing tool that

uses Cas9 as a nuclease guided by a single guide

RNA sequence to bind to a specified target DNA

sequence. Although CRISPR-Cas9 is primarily

driven by RNA-DNA interactions, there is one

remaining protein-DNA interaction known as the

protospacer adjacent motif (PAM). The PAM

sequence is required for Cas9 binding to a target

genomic site. Following Cas9 binding, a small DNA

R-loop is formed and a subsequent DNA double-

stranded break is generated by the Cas9

endonuclease.

One of the critical aspects of genome editing to

evaluate is the off-target propensity of each editing

agent. The guide RNA in Cas9 dictates a specific 20

base pair sequence of the targeted DNA sequence.

However, Cas9 binding can tolerate small

mismatches between the guide RNA and target DNA

sequence, meaning that there are possible binding

sites that are not the specified genomic site. Each

one of these undesired binding sites are known as

off-target sites in genome, which may cause

undesirable and dangerous negative effects that

could lead to additional genetic diseases and

unwanted phenotypes, such as cancer. Engineered

high-fidelity Cas9 variants have been developed as

an effective approach to decrease off-target effects.

SpCas9-HF1 is considered to be a high-fidelity

variant that maintains high on-target DNA editing

Precision Genome Editing towards the Treatment of Hemoglobinopathies

1067

efficiency (at least 70% of the wild-type SpCas9),

but dramatically reduced DNA off-target editing

efficiencies. The route of delivery into the cell also

has a role in the propensity of off-target editing.

Protein delivery further reduces off-target effects

compared to DNA viral or plasmid delivery since

DNA produces transcripts with subsequently product

a plethora of editor proteins. Furthermore, the life

span of DNA is longer compared to both RNA and

protein, which contributes to a longer exposure of

editor protein in cells. Because each cell only

contains a limited amount of DNA substrate for the

editing event, any additional editor protein has the

propensity to result in off target editing. Therefore,

delivering genome editing protein directly can

significantly decrease off target effects.

There are a variety of strategies developed

towards detecting Cas9 off target sites, which is vital

towards increasing the efficacy and safety of

CRISPR-Cas9 genome editing systems. At a single

cell level, whole genome sequencing delivers a

comprehensive view of the entire genome at a DNA

base level. However, off-target effects are

ineffective and occur at a relatively low percentage,

therefore requiring tens, hundreds, or even

thousands of single cell genome sequencing

evaluations to thoroughly evaluate all off-target

events. Therefore, it is expensive and not effective to

rely upon whole genome sequencing to thoroughly

evaluate the off-target propensity of every genome

editing agent. Alternative approaches to whole

genome sequencing were introduced that can enrich

for off-target editing events. These rely

fundamentally upon next-generation sequencing

techniques. GUIDE-seq (Genome-wide, Unbiased

Identification of DSBs Enable by Sequencing) and

CIRCLE-seq (Circularization for In vitro Reporting

of CLeavage Effects by sequencing) are two

methods that enrich for Cas9 off-target sites that

experience a DSB. There are computational methods

that can predict potential off-target sites in the

genome, but these computational methods are

deemed a preliminary and biased compared to

unbiased experimental approaches. GUIDE-seq

integrates a dsODN (double-stranded

oligodeoxynucleotide) donor template into cleavages

sites by non-homologous end joining repair inside a

cell’s genome directly. However, NHEJ is prone to

produce indels rather than incorporating exogenous

sequences, which suggests that the sensitivity of

GUIDE-seq is one aspect that still needs to be

considered. CIRCLE-seq is a highly sensitive in

vitro assay that serves as an alternative approach to

GUIDE-seq in detecting off target sites. CIRCLE-

seq relies on first generating and purifying libraries

of circularized genomic DNA, then treating this pool

of circles with Cas9 nucleases. Subsequently,

nuclease-linearized DNA fragments are ligated with

sequencing adapters and sequenced using next

generation sequencing to identify genome-wide

Cas9 nuclease off target sites.

Base editing is a relatively new genome editing

technology that is able to edit DNA without any

DSB intermediate. Base editing enables the ability to

perform chemistry directly on the genome of living

cells. Base editing fundamentally relies upon the

Cas9 component from the CRISPR-Cas systems

fused together with an enzyme that directly modifies

DNA or RNA. Dead Cas9 (dCas9) or nickase Cas9

(nCas9) is first guided by a sgRNA to the targeted

region in the genome. Following Cas protein

binding, the Cas protein exposes a single-stranded

DNA R-loop region that serves as substrate for

deamination mediated by deaminases fused to the

Cas protein. Base editing is currently largely

grouped into either cytosine BEs that can convert

cytosine-guanine (C-G) to thymine-adenine (T-A)

base pairs, or adenine BEs that can convert adenine-

thymine (A-T) to guanine-cytosine (G-C) base pairs.

Base editors create precise, predictable, and efficient

genetic outcomes at the targeted sequence without

any undesired indel byproducts or large genomic

perturbations such as p53 activation, DNA

rearrangements, or DNA translocations.

Similar to base editing, prime editing is able to

perform gene editing without inducing DSBs. But

prime editing can generate a wider range of possible

alterations as it can perform all twelve possible base-

to-base conversions. Prime editing is composed of a

Cas9 nickase fused to PE2 (a modified reverse-

transcriptase) and a multifunctional prime editing

guide RNA (pregRNA). A major advantage of prime

editing is its low off-target activity compared to

Cas9 as edition of PE2 significantly lowered the off-

target effects with fewer indels. Moreover, PE

breaks the bottleneck in therapeutic application of

gene editing as HDR machinery is not required for

prime editing so that post-mitotic cells can be edited.

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Table 1: Comparison of four commonly used genome editing biotechnologies.

Technology

Meganuclease ZFN TALEN CRISPR/Cas9

Origin

Microbial

genetic

elements

Eukaryotic gene

regulators

Bacterium

Xanthomonas

Adaptive immune

system in archaea and

bacteria

Targeting

Protein – DNA

interaction

Protein – DNA

interaction

Protein – DNA

interaction

RNA – DNA

interaction

Specifi

cDNA binding

elements

Tripletconfined zinc

finger proteins

Single-base

recoognition TALE

proteins

sgRNA

Off-target Effect

Low High Moderate Variable

Delivery

Major vector

systems

Major vector

systems

DNA, mRNA,

adenovirus, AAV

DNA, mRNA, viral

vectors with sufficient

packaging capacity

Time

Long (7-15 days)

Relatively long (5-7

days)

Short (1-3 days)

Targeting

Efficiency

Low Moderate Moderate High

Size

1kb*2 ~3kb*2

4.2kb (Cas9) +

1kb(RNA)

4 INITIAL USES OF DNA DSB

CUTTING FOR TREATMENT

Bone marrow and stem cell transplants are currently

the primary route of treatment for sickle cell disease

and β-thalassemia. In the last decade, the profound

discovery and development of genome editing

technologies has enabled the ability to use nucleases

that create a DNA double stranded breaks (DSBs) in

a specific region of the genome to be considered as

an initial and effective treatment of β-

hemoglobinopathies.

Genes that encode for the β-globin and α-globin

are located on chromosomes 11 and 16 in the human

genome, respectively. Embryonic hemoglobin, fetal

hemoglobin, and adult hemoglobin are three variants

of the hemoglobin protein expressed on the

erythrocyte at different times during development.

Fetal hemoglobin (HbF) is a tetramer consisting of

two α-globin chains and two β- globin chains.

Following the first three months post-conception, the

embryonic globin experiences a dramatic decrease in

globin synthesis, while the level of fetal hemoglobin

rises rapidly, serving as the primary form of

hemoglobin expressed in the fetus while in utero.

After the infant is delivered, the level of fetal

hemoglobin drops significantly, while the expression

of adult hemoglobin becomes the dominant form of

hemoglobin.

A regulatory region named as the locus control

region, LCR, regulates the expression of the encoded

globin genes, and is located 40 to 60 Kb upstream of

the beta-globin locus. BCL11A is a protein

expressed on the LCR region that binds to two

regions on the hemoglobin locus, HBG2 and HBG1,

which represses fetal hemoglobin expression,

signaling the production of adult globin genes. As

the mutation affecting sickle cell is located in the

adult globin gene, many therapeutic options being

explored using genome editing rely upon

upregulation and reactivation of fetal hemoglobin

expression to compensate for the sickled adult

globin gene. A reasonable approach to elevate the

amount of fetal hemoglobin expressed is to reduce

the BCL11A repressor activity. Certain mutations

found in the erythroid enhancer region can naturally

repress the amount of BCL11A being translated,

which ameliorates disease symptoms through fetal

hemoglobin compensation. Increased expression of

fetal hemoglobin is a sufficient at alleviating

symptoms of sickle cell because the fetal

hemoglobin has a similar to higher oxygen

saturation compared to that of adult hemoglobin. It

is encouraging that ongoing clinical trials have also

demonstrated that the induction of HbF can

ameliorate symptoms of sickle cell disease and β-

thalassemia.

Precision Genome Editing towards the Treatment of Hemoglobinopathies

1069

Figure 1: Visual Representation of the Mechanism of Deleting the BCL11A gene.

CRISPR-Cas9 gene editing is helping to tackle

sickle-cell disease in many ways. Firstly, Cas9

enzyme can be introduced with a guide RNA to

target and repair the faulty β-globin gene through

HDR replacement using an exogenous donor

sequence as the template to correct the disease-

causing mutation. The correction rate ranges from

7% to 50% depending on the editing tool used, such

as ZFNs, TALENs, and CRISPR/Cas9, and the

delivery method employed when delivering these

tools into induced pluripotent stem cell (iPSCs) and

HSPCs in vitro. However, xenotransplantation

experiments revealed that HDR lacks the generation

of long-term engraftable HSCs, shown by a 10%

drop in gene correction in vivo. This is a major

limitation to this direct replacement strategy so other

approaches are needed. Alternatively, a Cas9

enzyme can delete the gene encoding the BCL11A

repressors to increase the production of HbF. Like

previously discussed, fetal hemoglobin serves as a

sufficient replacement of adult globin in alleviating

sickle cell disease symptoms. Although these

approaches have been demonstrated to be effective,

the biggest drawback to using CRISPR-Cas9

nuclease is the creation of double-stranded breaks.

These DSBs have the potential to result in large

cellular perturbations such as chromathripsis,

activation of p53 pathways, and increased cell stress

signaling. Therefore, there is an urgent need for

alternative approaches of using genome editing

without any double-strand DNA break

intermediates.

5 PRECISION EDITING USING

BASE EDITING, PRIME

EDITING

Contrary to CRISPR-Cas9 nuclease editing, base

editing can generate direct conversion of one base

pair to another at a target site without any double-

stranded break intermediates. The DSB-free base

and prime editing systems ensure a superior safety

aspect of genetic modifications related to the

therapeutic treatment for β -hemoglobinopathies.

Two major classes of base editing currently exist: A-

to-G base editor (ABE) and C-to-T base editor

(CBE) that both are mediated by a deamination

reaction of either adenine or cytosine, respectively.

Base editing has the potential to be one of the most

superior gene editing technologies.

Two approaches employing base editing are

being explored in treating sickle cell disease: fetal

hemoglobin activation and direct correction of the

sickle-causing mutation. The first approach relies

upon recreating a phenomenon known as hereditary

persistence of fetal hemoglobin (HPFH) with base

editing. HPFH is a benign genetic condition

reflective of high levels of HbF expression in adults,

caused by either large deletions or point mutations in

the globin locus.

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Figure 2: Levels of HbF y-globin protein detected in

unedited and edited erythroid cells.

ABE can induce mutations found in HPFH on

the fetal hemoglobin genes, HBG1/2, without

generating any double-stranded break intermediates

or undesired cellular perturbation side effects.

Significantly, over 80% base editing was achieved at

the HBG1/2 promoters, and this translated to a 60%

increase in the expression of fetal hemoglobin in red

blood cells. Moreover, high levels of editing and

robust HbF induction are maintained after a long-

term in vivo engraftment study in mice.

Figure 3: Level of HbS sickle protein detected in unedited

and edited erythroid cells.

Figure 4: Level of y-globin protein levels in erythroid cells

after 16 weeks of editing.

After 16 weeks, 90% base editing at the HBG1/2

promotors was maintained in the erythroid and more

than 65% of the cells expressed fetal globin. The

second approach in using base editing for sickle cell

is the creation of the Makassar mutation. The

Makassar variant is a naturally occurring mutation at

the sickle site where beta globin has a glutamine

instead of alanine. This variant can be found in a

portion of the asymptomatic human population in

Northern Europe, suggesting that it is an alternative

beta globin genotype that maintains life symptom-

free. The Makassar mutation allows the β-globin to

function normally, which is an important discovery

because the sickle cell mutation requires a base edit

that converts adenine to thymine, a conversion from

purine to pyrimidine that is not currently possible

with existing technologies. The Makassar variant

relies upon an adenine to guanine mutation at the

sickle site, which can be achieved using an adenine

base editor. Experimental data showed that 80% of

sickle cells can be corrected into this Makassar

variant using ABE in cells from a sickle patient.

Studies demonstrate that the Makassar mutation is

successful in the elimination of the HbS globin. In

untreated controls, 100% of cells are classified as

sickle cells; in contrast, just 80% successful base

editing at the targeted region reduced the amount of

sickle cells to only about 10% of the total

population.

0

10

20

30

40

50

60

70

Unedited Edited

y-globin/total β-globin (%)

Levels of HbF gamma

globin protein

0

10

20

30

40

50

60

70

80

90

100

Unedited Edited

βs-globin/Total β -like globin (%)

Levels of HbS sickle

protein

0

10

20

30

40

50

60

70

Unedited Edited

y-globin/Total β-like globins (%)

y-globin protein

levels in erythroid

cells

Precision Genome Editing towards the Treatment of Hemoglobinopathies

1071

6 TARGET CELL TYPES AND

DELIVERY METHODS

The delivery of genome editing agents to desired

cell types is a grand challenge achieving precise and

effective gene editing. The molecular weight of

Cas9 protein 160kDa and the phosphate backbone of

the sgRNA creates an overall negative charge to the

Cas9 complex. There are two main pathways being

employed for Cas9 delivery: in vivo strategies and

ex vivo strategies. Each of these two approaches has

its unique advantages and disadvantages so they

each can be used in different circumstances.

Electroporation is a physical delivery method being

explored ex vivo. Electrical currents are used to

stimulate cells to create an instantaneous opening of

pores in the cell membrane to make it permeable and

enable the delivery of surrounding substrates.

Electroporation is widely used in ex vivo gene

editing since it is capable of being applied to a wide

range of cell types. However, the electrical current

generated by electroporation results in a high

percentage of cell death so it is difficult to scale this

method for widespread adaptation.

Lipid delivery is another commonly used

delivery approach relying upon the formation of

lipid nanoparticles to encapsulate substrate cargo.

Lipids are comprised of a hydrophilic polar head and

a long hydrophobic nonpolar tail. The encapsulation

of negatively charged nucleic acids into positively

charged liposomes aids in the entry of these

nanoparticles into cells through cellular endocytosis.

Once in the cell, the nanoparticle is degraded upon

traveling to late endosomes, which releases the

cargo and permits downstream genome editing.

Previous efforts have demonstrated that these lipid

nanoparticles can effectively package DNA, RNA,

and ribonucleoprotein complexes. Furthermore,

studies have demonstrated that direct injection of

these nanoparticles enable effective delivery into the

liver of living animals.

Viral vectors are another effective approach

being explored for gene delivery due to the

ubiquitous nature of viruses in the world. Most

commonly used viruses in research include the

adeno-associated viruses (AAVs), lentivirus, and

adenovirus. AAVs serve as a main vector to deliver

Cas9 to differentiated tissues by transduction.

Several clinical trials have been approved using

AAVs, which are less immunogenic compared with

other viruses. Although AAVs are effective at

delivery transgenes into cells in animals and

humans, a grand challenge for AAVs is the

limitation on the carrying capacity. AAV vectors are

typically limited to genes smaller than 4.8 kb, so any

gene larger would be unable to be delivered using

one AAV vector. The delivery of Streptococcus

pyogenes Cas9 (SpCas9) by AAVs is challenging

due to its large size of 4.2 kb, while the delivery of

Staphylococcus aureus Cas9 (SaCas9) is a more

feasible approach with a size of 3.15 kb. In addition

to the Cas protein, one would also need to package

the sgRNA so it is difficult to package everyone

onto one AAV vector. Previous studies have used

two AAVs to deliver an SpCas9 (4.8kb) and a

sgRNA (3.0kb) separately into a mouse brain to

target a gene called Mecp2. Furthermore, they

demonstrated that they could include two additional

sgRNAs into the virus and obtain multiple edits in a

single cell. In a subsequent study, they describe that

SaCas9, about 1kb smaller than SpCas9, and its

sgRNA can fit one a single AAV vector with a size

of 4.7kb and demonstrate that this single AAV

vector can be delivered into mice liver in vivo.

Lentivirus (LV) is another type of viral vector

used to deliver CRISPR-Cas9 ex vivo. LV vectors

have a more generous packaging capacity of 8kb,

which allows for including both a Cas9 protein and a

targeting sgRNA into a single LV vector. Lentiviral

vectors are mainly used in ex vivo gene delivery and

are currently used as the delivery vector in FDA-

approved chimeric antigen receptor-T therapies.

These approaches have shown superior delivery

efficacy in hematopoietic stem cells and T cells.

However, the limitation of the scope of targets for

LV is larger than AAV, and previous studies have

limited any in vivo delivery prospects of using LVs

in preclinical trials. Another challenge to using LVs

is the genotoxicity and immunogenicity defects.

However, integrase-defective lentiviral vectors

(IDLVs) have been introduced to allow for efficient

and continual transgene expression in vivo while

minimizing any undesired cellular effects.

Adenoviruses (AdVs) are widely used in clinical

trials for gene delivery as AVs are able to transduce

both dividing and nondividing cells. The genome of

AdVs generally range from 34 – 43 kb long and

AdVs do not integrate into host cell genomes, which

minimizes any potential off-target effects. However,

a major concern of using AdVs as a delivery method

is that AdVs trigger intense immune responses,

which leads to significant inflammation.

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Table 2: Delivery Method Comparison Chart.

Method Delivery Material Apprpach

Packaging

Capacity

Advantages

Disadvantages

Adenoviruses

Double-stranded

DNA

In vivo 8-30kb

1.High transfection

efficienc

2.Transduce both

dividing and non-

diving cells

High

immunogenicity

Adeno- associated

viruses

Single-stranded

DNA

In vivo <4.8kb

1.Low

immunogenicity

2.High

transfection

efficienc

y

Limited

packaging

capacity

Lentiviruses

Single-stranded

RNA

In vivo 8kb

1.High transfection

efficienc

2.Decent

packaging capacity

Potential

genotoxicity

Electroporation

DNA plasmid,

mRNA, RNP

Ex vivo -

1.Viralfree2.Fast

3High transfection

efficiency

Lowcell viability

Expensive

Lipid Nanoparticles

DNA plasmid,

mRNA, RNP

Ex vivo -

1.Viral free

2.Cheap

High toxicity

A major challenge to using viruses to deliver

genome editing agents is that many newer precision

genomes editing approaches are larger than the

original Cas9 system. For instance, if using base

editing, the fusion of a deaminase with Cas9

enlarges the size of the overall editor complex. A

recent study demonstrate that it is possible to split

the base editor in half and linking them together

using an intein system. Each half could be packaged

into two separate AAV systems and can reconstitute

into a full-length base editor upon delivery into a

target cell. This system was used to demonstrate

successful base editing in a mouse brain, and as a

result, in both cortex and cerebellum, about 50% of

base editing was observed in the targeted cells.

Although most efforts for treating

β−globinopathies rely upon ex vivo approaches, a

recent study explored the possibility of in vivo

genome editing. It is known that editing the

autologous HSCs demonstrate a prolonged benefit in

treating SCD. Ex vivo delivery of genome editing

agents for hemoglobinopathies begin by taking out

the stem cells of patients to perform gene editing in

the lab, and then reintroduce the edited stem cells

back to the patient. A significant limitation is when

delivering the edited cells back to the patient, the

patients need undergo myeloablation, which is a

process of significantly weakening the natural

immune response edited stem cells can survive and

engraft in the patient. This process in patients can

generate other types of detrimental disorders and be

very damaging to the patient’s overall health. In new

data released by Intellia Therapeutics, they

demonstrate that certain LNPs can deliver CRISPR-

Cas9 mRNA into hematopoietic cells direct in vivo.

Lipid nanoparticle with the Cas9 mRNA and the

target gRNA were delivered into the cells, and they

observed editing in the stem cells of animals treated

with these engineered LNPs. Increased editing was

observed with repeat multi-dosing. Intellia achieved

therapeutic levels of editing in human CD34+ cells

in a xenotransplanted mouse. Overall, this data

suggests that using engineered LNPs is a safe and

effective process to deliver genome editing agents

directly into animals with minimal side effect.

7 DISCUSS CLINICAL TRIALS IN

PROGRESS

There are many approaches in which genome editing

is being explored for the treatment of

hemoglobinopathies. In this final section, I will

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1073

discuss ongoing clinical trials conducted by Editas

Medicine, Beam Therapeutics, CRISPR

Therapeutics and Vertex Pharmaceuticals.

CRISPR-Cas9 mediated nuclease genome editing

through double-strand break intermediates is

considered as an initial and effective approach to

treat hemoglobinopathies. EDIT-301, introduced by

EDITAS, is an approach of editing HBG1/2 to

increase fetal hemoglobin expression as a

compensatory mechanism for sickled adult globin.

Key regulatory regions in the β-globin locus are

shown to be edited by SpCas9 and Cas12a. They

demonstrate that Cas9 and Cas12a editing allows for

a durable maintenance of indels at the target site.

After editing the HBG1/2 region, they demonstrate

that there is a constant amount of erythroid and

caspases produced, and there is no significant

increase in cell death. They evaluated that stem

blood cells treated with HBG1/2 editing displayed a

52% increase in the expression of fetal hemoglobin,

and that 89% of red blood cells will carry the fetal

hemoglobin compared to only 4% of red blood cells

when unedited.

Beam Therapeutics is a biotechnology company

that uses base editing as a therapeutic approach to

treat patients suffering from serious diseases. Beam

Therapeutics is exploring two uses of base editing

towards the treatment of hemoglobinopathies. First,

they use base editors to induce single base changes

in the regulatory regions of HBG1 and HBG2 to

disrupt repressor binding binds, which results in an

increased expression of fetal hemoglobin (HbF).

Second, they are exploring the use of adenine base

editing to directly edit the adenine implicated in

sickle cell disease to correct the E6V mutation into a

glutamic acid to reflect the Makassar variant. Their

initial data demonstrates that the Makassar program

is able to achieve 0% to 70% direct editing of the

sickle cell point mutation, which is sufficient

towards the alleviation of sickle cell symptoms.

A collaborative clinical trial between CRISPR

Therapeutics and Vertex Pharmaceuticals has

released preliminary findings on their therapeutic

program, CTX001, that targets BCL11A to result in

increased fetal hemoglobin expression. They

demonstrate that by editing patients’ own blood stem

cells with CRISPR-Cas9, they can achieve elevated

levels of HbF in red blood cells. The most recent

data reflect that five patients with beta thalassemia

and two patients with sickle cell disease treated

under CTX001 all have experienced successful

engraftment of edited blood stem cells and that they

had no vaso-occlusive crises (VOCs) during the

follow-up after the CTX001 infusion. Importantly,

all patients maintained near-normal hemoglobin

levels and showed drastic alleviation of

hemoglobinopathy symptoms.

It is exciting to continue witnessing the rapid

development of genome editing towards the

treatment of detrimental human disorders.

8 CONCLUSIONS

The rapid development of new genetic therapies

support a prosperous future for curing intractable

genetic disease. In the last few years, we have

witnessed the development of new technologies,

experimental models, and pre-clinical and clinical

studies. Insights into mechanism of action

demonstrated many possibilities of editing the β–

globin gene to introduce effective therapeutic

strategies that treat β–hemoglobinopathies.

While many encouraging clinical trials have been

released, many challenges still exist until we can

fully appreciate the full potential of these approaches

for curing these blood disorders. Firstly, the off-

target effects on DNA can potentially cause

irreversible damages, such as large genomic

rearrangements. Thus, there is a need to carefully

monitor these undesired events in clinical trials to

precisely plan for and adjust for any detected effects.

Specifically, the off-target activity of BEs, including

both sgRNA-independent and sgRNA-dependent

events require close monitoring. Fortunately,

engineering modifications into the deaminase have

lowered sgRNA-independent DNA off-target

activity while maintaining highly efficient on-target

DNA editing. Secondly, even though BE and PE

have overcome cytotoxicity events caused by DSBs-

induced indels, proper delivery methods are still

needed, especially for primary cells. However, it

remains a challenge to develop efficient methods

that can deliver the large complex sizes of BE and

PE technologies. Thirdly, the current pool of BEs

only enable C-T, C-G, and A-G conversions;

therefore, more optimizations and technologies are

required to enable other types of conversions. Prime

editing is a new approach that can overcome many

of the editing types possible; however, further

optimizations are needed to realize higher editing

outcomes. Despite these challenges, gene and cell

therapy hold great promise for providing proper

treatment approaches for patients diagnosed with β–

hemoglobinopathies.

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ACKNOWLEDGEMENTS

If any, should be placed before the references

section without numbering.

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