gene therapy, procedure, application and approaches

34. 03-04-2025

35. ex vivo gene therapy
Ex vivo gene therapy is a technique where a patient's cells are
modified outside of their body and then transplanted back into
them. Here's a breakdown of key aspects:
Key Concepts:
•Process:
• Cells, often blood stem cells, are removed from the
patient.
• In a laboratory, genes are introduced or modified within
these cells.
• The modified cells are then returned to the patient.
•Purpose:
• To correct genetic defects or equip cells with new
functions, such as enhanced cancer-fighting abilities.
• It's often used for conditions affecting blood and bone
marrow.

36. ex vivo gene therapy
•Advantages:
• Greater control over gene modification.
• Allows for quality control and verification of the modified
cells before they are returned to the patient.
• Can reduce the risk of unwanted immune responses.
•Examples:
• CAR-T cell therapy for certain cancers.
• Gene therapies for blood disorders like sickle cell disease
and beta-thalassemia.
Key Differences from InVivo GeneTherapy:
•In vivo gene therapy involves directly introducing genetic
material into the patient's body, while ex vivo gene therapy
modifies cells outside the body.

37. L42 : In-vivo gene
therapy
In vivo gene therapy involves directly introducing genetic
material into a patient's body to modify cells and treat disease.
Here's a breakdown:
Key Concepts:
•Direct Delivery:
•Unlike ex vivo gene therapy, where cells are modified
outside the body, in vivo therapy delivers therapeutic genes
directly into the patient.
•This is often achieved using vectors, most commonly
modified viruses, which carry the genetic material to target
cells.
•Mechanism:
•The introduced genes aim to replace defective genes,
introduce new genes, or modify gene expression to achieve a
therapeutic effect.
•Delivery methods include intravenous injection, direct
injection into specific tissues, or inhalation.

38. L42 : In-vivo gene
therapy
•Applications:
•In vivo gene therapy is being explored for a
wide range of conditions, including:
•Inherited genetic disorders.
•Cancer.
•Neurological diseases.
•Eye diseases.
In essence, in vivo gene therapy offers the
potential to treat diseases by directly modifying
a patient's genetic makeup within their body.

39. L42 : In-vivo gene
therapy
•Vectors:
•Viral vectors, such as adeno-
associated viruses (AAVs), are
commonly used due to their
ability to efficiently deliver
genes1
into cells.
•Non-viral vectors, such as lipid
nanoparticles, are also being
developed.

40. Key Considerations:
•Targeting:
•Ensuring that the therapeutic genes reach the
intended target cells is crucial.
•This can be challenging, as vectors may affect
non-target tissues.
•Immune Response:
•The body's immune system may react to the
vector or the introduced genes, potentially
causing adverse effects.
•Long-Term Effects:
•The long-term safety and efficacy of in vivo
gene therapy are still being studied.

41. Approaches Used
in gene therapy

42. Target cells of gene therapy
Factors Determining Target Cells:
•Disease Location:
•The location of the disease dictates which cells need to
be targeted. For example:
•Eye diseases: Retinal cells.
•Neurological disorders: Neurons in the brain or spinal
cord.
•Liver diseases: Liver cells (hepatocytes).
•Blood disorders: Hematopoietic stem cells in the bone
marrow.
•Cell Accessibility:
•Some cells are more easily accessible than others. For
example, blood cells can be readily extracted, while brain
cells are more challenging to reach.

43. Target cells of gene therapy
•Cell Type and Function:
•The specific function of the target cells is also
important. For instance:
•Immune cells (T cells) are targeted in CAR-T cell
therapy for cancer.
•Muscle cells are targeted in gene therapies for
muscular dystrophy.
•Vector Capabilities:
•The type of vector used to deliver the therapeutic
gene influences which cells can be targeted.
Different vectors have different cell tropisms
(preferences).

44. Target cells of gene therapy
•Hematopoietic Stem Cells
(HSCs):
These are targeted in gene
therapies for blood disorders
like sickle cell disease and
hemophilia.
•T Cells:
These are targeted in CAR-T
cell therapy for certain cancers.
•Retinal Cells:
These are targeted in gene
therapies for inherited retinal
diseases.
•Muscle Cells:
• These are targeted in gene
therapies for muscular
dystrophies.
•Liver Cells (Hepatocytes):
• These are targeted in gene
therapies for some
metabolic diseases.
•Neurons:
• These are targeted in gene
therapies for some
neurological disorders.

45. Approaches
Used
in
gene
therapy

46. Brief about the
viral gene transfer

47. •Viral Vectors:
•Modified viruses, known as viral vectors, are used
as delivery vehicles.
•These viruses are genetically engineered to
remove their disease-causing components, making
them safe for use.
•The therapeutic gene is then inserted into the viral
vector.
•The process by which a viral vector introduces its
genetic material into a host cell is called
transduction.
•The viral vector binds to the target cell and
releases its genetic payload, which includes the
therapeutic gene.

48. •Types of Viral Vectors:
•Several types of viruses are used as vectors, each with its
own characteristics:
•Adeno-associated viruses (AAVs): These are safe and
efficient vectors that can deliver genes to a variety of cell
types.
•Adenoviruses: These vectors can carry larger genes
but may trigger an immune response.
•Retroviruses: These vectors integrate their genetic
material into the host cell's genome, providing long-term
gene expression.
•Lentiviruses: A subtype of retroviruses, these can also
integrate into the host cells genome, and can infect non-
dividing cells.
•Herpes simplex viruses (HSVs): These vectors are
often used for gene delivery to the nervous system.

49. Nonviral gene
transfer

50. deliver therapeutic genes into cells, bypassing the use of
viruses. This approach has gained significant attention
due to its potential for increased safety and versatility.
Here's a breakdown:
Key Characteristics:
•Safety:
•Nonviral vectors generally pose a lower risk of
triggering an immune response compared to viral
vectors.
•They also reduce the risk of insertional mutagenesis,
a concern associated with some viral vectors.
•Versatility:
•Nonviral methods can deliver a wider range of
genetic material, including large DNA sequences and
RNA.
•They can also be adapted to target various cell types.

51. Common Nonviral Gene Transfer Methods:
•Physical Methods:
•Electroporation:
•This technique uses electrical pulses to create temporary
pores in cell membranes, allowing genetic material to
enter.
•Gene Gun:
•This method propels DNA-coated particles into cells at
high speed.
•Microinjection:
•This involves directly injecting genetic material into cells
using a fine needle.
•Chemical Methods:
•Liposomes:
•These are lipid-based vesicles that encapsulate and deliver genetic material.
•Polymers:
•Synthetic polymers can form complexes with DNA or RNA, facilitating their
entry into cells.
•Nanoparticles:
•Tiny particles that can transport genes into cells. Lipid nanoparticles are a
very promissing type of non-viral vector.

52. Advantages:
• Reduced risk of immunogenicity.
• Large cargo capacity.
• Relatively easy to manufacture.
Disadvantages:
• Lower transfection efficiency compared to
some viral vectors.
• Often results in transient gene expression.
In summary, nonviral gene transfer offers a
promising alternative to viral vectors, with
a focus on safety and versatility.

53. pH-sensitive:
Nonviral gene
transfer

54. The Challenge: Endosomal Escape
• A major hurdle in nonviral gene transfer is that after the gene-
carrying vector enters the cell, it often gets trapped in structures
called endosomes.
• Endosomes are acidic compartments, and if the vector and its
genetic cargo remain trapped, they are likely to be degraded,
preventing the therapeutic gene from reaching the nucleus
where it needs to act.
pH-Sensitive Strategies:
• To overcome this, researchers have developed pH-sensitive
nonviral vectors that are designed to destabilize or disrupt
endosomal membranes when they encounter the acidic
environment within the endosome.
• This "endosomal escape" allows the therapeutic gene to be
released into the cytoplasm, increasing the chances of successful
gene expression.

55. How it Works:
•These vectors often incorporate materials that undergo
structural changes in response to low pH.
•For example:
• Certain polymers become more hydrophobic in acidic
environments, causing them to interact with and disrupt the
endosomal membrane.
• Lipid-based vectors can be designed with components that
become destabilized at low pH, facilitating membrane fusion
and release of the genetic material.
Significance:
•pH-sensitive nonviral gene transfer is a crucial area of research
because it addresses a key limitation of nonviral delivery systems.
•By improving endosomal escape, researchers aim to increase the
overall efficiency and effectiveness of nonviral gene therapy.

56. Cationic lipid liposomes strategy
ore Principles:
•Liposomes:
• Liposomes are spherical vesicles composed of lipid
bilayers. These structures can encapsulate various
substances, including DNA and RNA.
•Cationic Lipids:
• Cationic lipids are lipids with a positive charge. This
positive charge is crucial because it allows the liposomes
to:
• Interact with negatively charged nucleic acids (DNA,
RNA), forming complexes.
• Interact with the negatively charged cell membrane,
facilitating cellular uptake.

57. •Mechanism of Action: The positively charged
liposomes form complexes with negatively charged
nucleic acids.
•These complexes then interact with the cell
membrane, often through electrostatic interactions.
•The liposomes can then enter the cell via
endocytosis or, in some cases, by fusing with the
cell membrane.
•Once inside the cell, the goal is for the genetic
material to be released from the endosome and
reach the nucleus (for DNA) or cytoplasm (for
RNA).

58. Key Advantages:
•Safety:
• Compared to viral vectors, cationic liposomes
generally have a lower risk of triggering an immune
response.
•Versatility:
• They can be used to deliver various types of genetic
material.
• They can be modified to target specific cell types.
•Relatively easy to produce:
• Manufacturing these liposomes is generally less
complex than producing viral vectors.

59. Key Challenges:
•Endosomal Escape:
• A major challenge is getting the genetic material out
of the endosome after it enters the cell.
•Transfection Efficiency:
• The efficiency of gene transfer can be lower
compared to some viral vectors.
•Toxicity:
• Cationic lipids can sometimes exhibit toxicity.
• Interactions with serum proteins can reduce the
effectiveness of these liposomes.

60. Applications:
•Cationic liposomes are used in gene
therapy, drug delivery, and other
biomedical applications.
•They are particularly relevant for
delivering nucleic acids, such as DNA,
mRNA, and siRNA.