PHAR 203 - Gene Therapy

What is gene therapy?

a person’s genes are modified to treat or cure a disease by introducing foreign genetic material, such as RNA or DNA

Why develop gene therapies?

Rare genetic disorders have limited therapeutic options and lack early diagnosis, gene therapy treats these diseases at the root, and patients produce their own therapeutic gene product

What is the difference between enzyme replacement and gene augmentation

abnormal enzymes are non-functional and they’re treated by injecting purified enzyme into patient, which was produced by recombinant system

gene augmentation replace the mutant version with a new functional version that produces correct protein → healthy cell

There are two ways to deliver genetic material to patients, what are they?

In vivo and Ex vivo

What is in vivo way to deliver genetic material to patients

direct administration of a vector that has a genetic material

What is ex vivo way to deliver genetic material to patients

• extract patient cells

• genetic modification by a vector carrying a therapeutic transgene

• selection and expansion in culture

• infusion to reintroduce the engineered cells back into patient

Do Genetic Materials need a delivery vector and why?

yes, because nucleic acids are large and negatively charged, they poorly accumulate and penetrate into deeper tissues, biological enzymes rapidly degrade naked RNA / DNA molecules, and rapid clearance by immune cells.

What are the physical methods of gene delivery types (5)

• electroporation

• sonoporation

• microinjection

• gene gun

• optoporation

What are the non-viral methods of gene delivery (5)

• Lipid based Nanoparticles (LNPs)

• Lipid—Polymer Hybrid Nanoparticles

• Extracellular vesicles

• inorganic nanoparticles

• cationic nanoparticles

Gene gun (biolistics) delivery involved coating DNA onto metal particles and firing them onto tissue. Which metal is most commonly used, and why is particle size a critical parameter for this technique

Gold or tungsten particles are used; particle size must be 0.6-2 microm to penetrate the cell membrane

What are the viral methods of gene delivery types (4)

• retrovirus (8 kb)

• lentivirus (9-10 kb)

• AAV (adeno-associated virus) (4.7 kb)

• adenovirus (36 kb)

What is the expression of retrovirus

stable

What is the expression of lentivirus

stable

what is the expression of AAV

transient or stable

what is the expression adenovirus

transient

does the retrovirus have target cell genome integration

yes

does the lentivirus have target cell genome integration

yes

does the AAV have target cell genome integration

No

Recombinant AAV has low frequency of host genome integration

does the adenovirus have target cell genome integration

No

What are the characteristics of retrovirus

• high risk of insertional mutagenesis

• can only target dividing cells

what are the characteristics of lentivirus

• can trigger immune reaction

• can target dividing and non-dividing cells

what are the characteristics of AAV

• small cargo size, sustained expression

• need a helper lipid to infect

• can target dividing and non-dividing cells

what are characteristics of adenovirus

• highly immunogenic

• short expression duration / transient

• does not integrate with host genome

What is an AAV?

A small, non-pathogenic virus used as a vector in gene therapy

What is the size of AAV?

18-26 nm in diameter

What kind of genome does AAV contain?

• non enveloped

• icosahedral capsid

Does AAV cause disease?

No, AAV is non-pathogenic (doesn’t cause disease)

Can AAV replicate independently?

No, It needs a helper virus for replication

Which viruses help AAV replicate?

• adenovirus

• herpesvirus

• vaccinia virus

Why is AAV widely used in gene therapy?

Because it is:

• safe (non-pathogenic)

• low immune response

• can infect many cell types

What does it mean that AAV has a broad host range?

It can infect many different cell types, making it versatile

What are AAV serotypes

variants with different surface proteins, leading to different tissue targeting

Why are different AAV serotypes useful?

they allow targeting of specific tissues (tissue tropism)

What is tissue tropism?

the ability of virus to target specific cell types or tissues

What does minimal immunogenicity mean?

it causes little immune response, making it safer for therapy

Summarize AAV in one line

AAV is a safe, non-pathogenic ssDNA virus with broad host range and low immunogenicity, making it idea for gene therapy

Walk through AAV lysogenic lifecycle

• AAV binds receptor

• endocytosed into cell

• endosomal escape by degradation and transit to nucleus

• coat removal

• second strand DNA synthesis

• Integration into host genome (AAVS1 site on Chr 19)

• Viral DNA as Episomal DNA

Walk through AAV lytic lifecycle

• AAV binds to receptors

• endocytosed into cell

• endosomal escape

• coat removal

• second strand DNA synthesis

• Co-infection with helper virus (specific gene products needed to express AAV replication and capsid proteins)

• DNA replicates

• Capsid made

• virions assemble

• release

What is the first step in creating an AAV gene therapy vector

the viral genome is cloned

What is done to the original AAV viral genes

they are removed to prevent viral replication and pathogenicity

What is inserted into the AAV genome

the target (therapeutic) genes

why are viral genes removed from AAV

to make the virus safe and unable to replicate on its own

What is done after the recombinant DNA is created?

it is used to transform bacterial cells for amplification

Why are bacteria used in this process?

to replicate and produce large amounts of plasmid DNA

why are helper virus genes needed

they provide functions required for AAV replication and packaging

What is the plasmid used for in AAV gene therapy?

A DNA vector carrying:

• therapeutic gene

• necessary regulatory elements

What happens when AAV infects target cells

the DNA can integrate into the genome (or persist episomally)

What are recombinant AAV (rAAV) particles?

Engineered AAV viruses that carry the therapeutic gene

Can recombinant AAV replicate on its own?

No it can’t replicate independently

Why are rAAV vectors considered safe?

because they

• lac viral genes

• can’

Why is AAV called a biological nanoparticles?

Because it is a tiny, engineered particle used to deliver genes into cells

What is the overall process of AAV gene therapy

• clone AAV genome

• remove viral genes

• insert therapeutic gene

• amplify DNA in bacteria

• provide helper virus functions

• produce rAAV particles

• deliver gene to target cells

Which of the following best explains why AAV-based gene therapy is generally administered as a single lifetime dose, and what is the main obstacle to re-administation if a booster dose were needed?

AAV integrates into every cell of the target tissue simultaneously, providing permanent genetic correction.

3 Major Limitations of AAV-mediated Gene Therapy

• high cost of treatment

• high cost of viral vector production & purification

• ineffective re-administration due to vector - neutralizing antibodies

Which limitation is most characteristic of physical transfection methods when compared to chemical nanoparticle-based delivery?

A. Low transfection efficiency

B. High dependence on endocytic pathways

C. Reduced scalability and increased cell damage

D. Poor Nucleic acid protection from degradation

Nucleic acids are

genetic materials used for gene therapy

Nucleic acids have (3)

• high specificity

• functional diversity

• limited toxicity

What are polyplexes in gene delivery?

Complexes formed between cationic polymers and nucleic acids

Why are cationic polymers used in gene delivery?

They are positively charged, so they bind to negatively charged DNA/RNA

What is an example of cationic polymers used for gene delivery?

Diethylaminoethyl (DEAE)-dextran

Which cationic polymer is considered one of the most efficient for gene delivery?

Polyethyleneimine (PEI)

What is a major bottleneck of cationic polymers Gene delivery systems

toxicity and low efficiency

Why do toxicity and low efficiency limit clinical translation?

they reduce safety and effectiveness, making them less suitable for use in patients

What are recent advancements in cationic polymers design?

Development of

• biodegradable polymers

• bio-reducible polymers

• stimuli-responsive polymer

• targeted polymers

What is the goal of designing new types of cationic polymers

to reduce toxicity and improve delivery efficiency and specificity

What makes up the building block of living cells?

Lipids

What is a phase transition temperature (Tm) of phospholipids

the temperature that a lipid membrane goes from solid gel to fluid liquid

Tm (phase transition temperature of phospholipids) can be measured by

differential scanning calorimetry (DSC)

What happens to Tm (phase transition temperature of phospholipids) when there is an increased chain length → saturated chain

Tm increases

Lipid self assembly is governed by

molecular geometry and charge

Lipid shape depends on

• headgroup size and hydration

• fatty chain volume

• electrostatic interactions

Formula of Critical Packing Parameter (CPP)

Which factor would most increase the transition temperature (Tm) of a phospholipid

A. increasing the number of double bonds

B. shortening fatty acid chain length

C. Increasing saturation of fatty acid chains

D. Introducing cis double bonds

C. Increasing saturation of fatty acid chains

Liposomes are often consisting of

phospholipids and cholesterol

Describe liposomes

lipid bilayer made of ampipathic molecule surrounding an aqueous core, they’re neutral and has poor encapsulation

Describe the lipoplex

Lipoplex is formed by electrostatic interactions between positive and negatively charged nucleic acids, condenses into small particles when mixing.

What are the limitations of Lipoplexes

• aggregation with serum proteins

• cleared rapidly by immune cells

• unencapsulated nucleic acid = activated immune system

• less tolerability in vivo

3 examples of cationic lipids used in lipoplexes

1. DOTMA

2. DOTAP

3. DSTAP

Lipid nanoparticles are composed of 4 different components, each having a critical role in determining size, entrapment efficiency, stability and in vivo fate, what are these 4 components

1. ionizable cationic lipids

2. PEG lipid

3. Cholesterol

4. Helper lipids

Function of ionizable cationic lipids in LNPs

ionizable cationic lipids protonate at acidic pH, bind to nucleic acids and are neutral at physiological pH

Function of PEG Lipids in LNPs

PEG have stealth properties, minimize opsonization by serum proteins

Function of cholesterol in LNPs

cholesterol enhances stability and aid in transfection

Function of Helper lipids in LNPs

Helper Lipids promote cell binding, encapsulation efficiency and endosomal escape

Benefits of PEGylation (Shielding effect)

• increase particle stability

• increase circulation time

• decrease particle side

What does the Shielding effect of PEGylation do

the shielding effect can decrease cell uptake

Anti-PEG antibodies may lead to

reduced efficiency

Two LNP formulations have the same size, but only one results in high protein expression. What ionizable lipid property most likely explains this difference?

A. the charged ionizable headgroup

B. Length of the hydrophobic lipid tail

C. Optimized pKa and unsaturated tail

D. Increased carbon saturation in the tail

C. Optimized pKa and unsaturated tail

Because they escape endosome → high protein expression

First, the LNPs are tested on animal (Preclinical studies) - Human trials, what is the process when we wanna study about LDL

1. LNP administered IV to mouse

2. Accumulation of LNP in hepatocytes

3. Mechanistic study = show interaction between LNPs and LDL receptors

How nanoparticles are taken up inside cells

Endocytosis

• cell taking up LNPs (engulf)

What is the first step in LNP uptake after IV administration

rapid release of PEG-lipids from LNP particle

Why is PEG release important for LNP function

allows binding of ApoE to the LNP

What is ApoE

an exchangeable apolipoprotein found on lipid particles like VLDL (very-low-density lipoproteins)

What types of particles can ApoE bind to

• endogenous particles (lipoprotein)

• exogenous particles (LNPs)

What is Step 2 of LNP uptake?

Extravasation into the liver from circulation

What properties allow LNPs to enter the liver barrier?

• small size (<100nm)

• neutral charge

What happens in Step 3 of LNP uptake

ApoE-coated LNPs bind to LDL receptor on hepatocytes

What is the result of ApoE binding to LDL receptors

Receptor mediated endocytosis of LNPs

What cellular structure forms after LNP uptake?

endosome (intracellular vesicle)

What happens to pH inside the endosome?

it decreases (pH < 6.2) → becomes acidic

What happens to ionizable lipids in acidic endosomes?

they become protonated (positively charged)