The document summarizes a gene delivery system project submitted by two students. It describes how gene delivery systems provide a new perspective for modern medicine by allowing therapeutic genes to be inserted into target cells using viral or non-viral vectors. Viral vectors commonly used include adenoviruses, retroviruses, and adeno-associated viruses. Non-viral methods include using naked DNA, physical methods like electroporation, and chemical methods like lipoplexes and dendrimers. The conclusion states that gene delivery systems allow medicines to be directly injected into cells to cure diseases by attacking cells at the genetic level.

1. Bio Material Project
GENE DELIVERY SYSTEM
Submitted By:
Punith Kumar Neelam (WSU ID:M369K625)
Abhijith reddy Sadhu (WSU ID:M726Z747)

2. Abstract:
• Gene delivery system provided modern medicine a new
perspective which was unimaginable a few decades ago.
• Progress in the fields of molecular biology and r-DNA
technology is now changing the basis of clinical
medicine.
.
• Gene delivery system is currently employed in the
treatment of diseases like cystic fibrosis, cardiovascular
diseases, cancer and auto-immune diseases.

3. INTRODUCTION:
• Genes who are carried on chromosomes are basic
structural & functional units of heredity.
• Genes are specific sequences of bases that encode
instructions on how to make proteins.
• Genes get a lot of attention it’s the proteins that perform
most life function and form most of the cellular
structures.
.
• Genes are altered then the encoded proteins are unable
to carry out their normal functions which result in the
genetic disorders.

4. GENE DELIVERY:
• In most gene delivery studies a normal gene is inserted into the genome
to replace an abnormal disease causing gene.
• A carrier molecule called vector carries the therapeutic gene to patient’s
target cells. Vectors generally used are viruses.
• Target cells such as the Patient’s liver or lungs are infected with the viral
vector.
• The vector then unloads its genetic material containing the therapeutic
gene into the target cell..

5. Literature Review

6. VECTOR AND ITS IDEAL PROPERTIES:
A Vector can be described as a system fulfilling
several functions
1. Enabling delivery of genes to target cells
and their nucleus.
2. Providing protection from gene degradation.
3. Ensuring gene transcription in the cells.

7. VIRAL VECTORS:
• The viral vectors increase the capability of viruses to transfer genetic
material into the infected cell.
• Viruses insert their DNA into cells with high efficiency.
• Vectors are evolved by genetic modification of
retroviruses, adenovirus, adeno-associated virus & Herpes simplex virus.

8. ADENOVIRUSES:
• The Adenovirus genome comprises of a double stranded DNA molecule
which is linear with a diameter of 70mm.
• These are the best and mostly used systems for gene transfer.
• Three generations of adenoviral vectors have been developed depending on
the time course of expression during viral replicative cycle.

9. RETROVIRUSES:
• These are the first constructed Human gene therapy vectors.
Retrovirus is an enveloped virus particle.
• It contains two copies of viral RNA genome, rounded by a cone
shaped core. The viral RNA contains three essential genes
gag, pol, and env.
• gag→ This encodes core proteins, capsid, matrix and nucleocapsid
which are evolved by proteolysis cleavage of the gag precursor
protein.

10. RETROVIRUSES (Contd.)
• pol→This encodes for viral enzyme protease, reverse transcription and
integrate which are derived from gag-pol precursor.
• env→This encodes for envelope glycoprotein which encodes envelope
for glycoprotein which carry out arrival of virus.

11. ADENO ASSOCIATED VIRUSES (AAV):
• Adeno associated is a small virus infects humans and some other primate
species i.e. is not death causing virus which helps for immune response and
also used in gene therapy and AAV viruses to the parvoviruses.
• Generally it is 20nm long and 4.5kb size non-enveloped DNA viruses.
• It is a single stranded DNA which causes latent infection to human cells.
Parvoviruses gives alternative to malignancy- related retroviruses.

12. Structure of AAV:
1. AAV Genome: Genome built of SSDNA (Single Stranded
deoxyribonucleic) in positive or negative sense and it consist of ITRs
(Inverted terminal repeats) on both ends of SSDNA and two reading
frames (ORFs).
2. Inverted terminal repeater Sequences: ITRs are named because there
are symmetrical in nature and these are 145bases each. The important
property of this is it form hairpin which are useful in self primase
which are independent synthesis of the second DNA strand.
3. Rep genes : It one of the open reading frame on the left side of the
genome with different lengths
4. Cap genes: This is another reading frame we found in genome which is
generally on the right hand side.
5. VP Proteins: VP Proteins are the part of the Cap genes generally these
are 3 in number named as V1, V2, and V3.

13. Structure of AAV (Contd)
Infection Cycle of AAV:
 Cell membrane attachment.
 Endocytosis.
 Trafficking of endosomal
 Late endosome or lysosome escape
 Nucleus translocation
Advantages:
1. AAV apparent lack of pathogenicity which makes it better than retro
viruses.
2. This can be used in cancer treatment
Disadvantages:
1. Limited cloning capacity of the vector.
2. AAV ITR of two genomes is almost double the capacity of the
vector.

14. Herpes Simplex Virus (HSV):
An enveloped, double-stranded DNA virus with the size 150 kb is Herpes
Simplex Virus. HSV is a natural human pathogen and replicating in
epithelial cells. There are two members in Herpes family that can infect the
human body.
Herpes simplex virus structure:

15. Herpes Simplex Virus (HSV) (Contd).
Gene Vectors can make from the HSV in 2 methods:
Gene Vectors can make from the HSV in 2 methods:
1. Cloning therapeutic gene into plasmid which contains HSV and can be
packed in the cells and infects with the help of HSV.
2. Cloning gene into a plasmid which was surrounded by HSV sequences and
co transfected to cells with HSV.

16. Herpes Simplex Virus (HSV):
Advantage:
1. HSV Based Gene Therapy has the largest cloning capacity than all
the other.
2. The HSV genome is the largest of all the Viral vectors
Limitations:
HSV turns off all the expression of gens so only small portion of HSV
genome will active during latency. This can overcome by introducing
foreign gene into the latency region.

17. Non-Viral delivery Systems:
Non-viral delivery system has some advantages over viral system. Non-viral
are simple and safer alternative for the viral systems and low host
immunogenicity will be two.

18. Naturally Occurring compounds:
It is simplest method of injecting naked DNA into the human body i.e. it also
called Naked DNA. In this method naked plasmid DNA was used for injecting
intramuscular which is secured and relatively low level of expression and but
sufficient for use in DNA vaccination.

19. Naturally Occurring compounds:
Advantages:
1. Unlimited virtual clone capacity
2. Low Toxicity
3. Immunogenicity of non-viral vectors
Disadvantage:
1. Low transfection efficiency
2. Short term expression.

20. Enhancing of drug delivery by Physical methods:
There are 4 Physical methods by which we can enhance our drug
delivery they are:
• Electroporation
• Gene Gun
• Sonoporation
• Magnetofection

21. Electroporation:
• In this method we use short pulses of high
voltage to carry DNA across the cell
membrane which makes a shock to cause
temporary formation of pores and thus allow
DNA molecules to pass.
• Some of the drawbacks of electroporation
can be overcome by using of high-voltage
plasma discharge DNA was efficiently
delivered following very short pulses.

22. Gene Gun:
• In this method DNA is
coated with gold particles
and loaded into a device
• which is similar to gun
and it generates force by
which it can penetrate
into the cell.

23. Sonoporation:
We use ultrasonic frequencies to deliver DNA into
cells. Which can disrupt the cell membrane and allow
DNA to move into cells.

24. Magnetofection:
DNA is made into magnetic particles, and a magnet is placed
underneath the tissue culture dish to bring DNA complexes
into contact with a cell monolayer.

25. Enhancing of Drug delivery by Chemical Method:
There are 4 Chemical methods by which we can enhance our drug
delivery they are:
• Oligonucleotides
• Lipoplexes
• Dendrimers
• Inorganic Nanoparticles

26. Oligonucleotides:
In gene therapy oligonucleotides is to deactivate the genes involved as
a part of disease process. It involves two methods.
1. Using antisense to specific target gene and to disrupt the
transcription of the faulty gene.
2. By using small molecules of RNA for signal the cell and to cleave
specific unique sequences in the mRNA.

27. Lipoplexes:
For curing cancer we use lipoplexes most commonly. We use cationic
lips which are positively charged used to condense negatively charged
DNA molecules so as to facilitate the encapsulation of DNA into
liposomes.

28. Dendrimers:
A spherical shape highly branched macromolecules . The surface of
the particle may be functionalized in many ways and many of the
properties of the resulting construct are determined by its surface. It
has some limitations but even though it is considered as the one of the
best method in non-viral drug delivery.

29. Inorganic Nanoparticles:
It one of the non-viral gene delivery system where we use gold .silica
and iron oxide and calcium phosphate some of the benefits are storage
stability, low manufacturing cost and often time, low immunogenicity.

30. Hybrid Methods:
There is another method other than viral and non-viral gene delivery
system i.e. Hybrid gene delivery which is developed from the
combination of two or more techniques. One of the hybrid methods
is virosomeswhich is formed by the combination of liposomes with
activated HIV

31. Conclusion:
• Gene therapy utilizes the delivery of DNA into cells, which can be
done by several ways by a number of methods that is some of them
are by viral methods and some are by the non-viral methods few
are by the Hybrid methods.
• When a disease attack the human the virus will attack the cell of the
body instead giving medicines to the body or through blood if we
can inject them directly to the cell we have better chances of curing
disease.
• By using gene delivery system we are able to inject the medicine
directly to the cell so these drugs will work on the cell and we can
cure many more disease since they affect the human immune
system by attacking the cells.

32. Bibliography:
1. Brisson M, He Y, Li Setal: A novel T7 RNA polymerase autogene
for efficient cytoplasmic expression of target genes.
2. Darquet AM, Rangara R, Kiers P et al: Minicircle: an improved
DNA molecule for in vitro and in vivo gene transfer.
3. NONTEMPLATE-DEPENDENT POLYMERIZATION PROCESSES:
POLYHYDROXYALKANOATE SYNTHASES AS A PARADIGM, JoAnne
Stubbe, Jiamin Tian, Aimin He, Anthony J. Sinskey, Adam G.
Lawrence, and Pinghua Liu.