Gene Therapy

Presented by-

Panchali Das

M.Sc. Biotechnology

4th Semester

BBAU

Knowing more with the help of

Retrovirus-Lentivirus

Retrovirus

• A retrovirus is a single-stranded positive sense RNA virus that

replicate via a double-stranded DNA intermediate.

• It is roughly 100 nanometers in diameter.

• The term "retro" in retrovirus refers to the reversal of the central

dogma of molecular biology.

• It is one of the mainstays of current gene therapy approaches.

Retroviruses as vectors

• Certain retroviruses are acutely oncogenic.

• Investigation has shown that the viral oncogenes are proto-oncogenes.

• The viral oncogenes are found to be expressed as fusions with essential viral genes, rendering the virus replication defective.

• Most retroviruses do not kill the host, but produce progeny virions over an indefinite period.

• Retroviral vectors can therefore be used to make stably transformed cell lines (undergo phenotypic change).

Retroviruses as vectors

• Viral gene expression is driven by strong promoters, which can be subverted to control the expression of transgenes.

• Murine mammary tumor virus

• Have a broad host range allowing the transduction of many cell types.

• E.g. amphotropic strains of murine leukemia virus (MLV)

• Make efficient and convenient vectors for gene transfer because the genome is small enough for DNA copies to be manipulated in vitro in plasmid-cloning vectors..

Inducible by glucocorticoids

(Transcription)

Replication & Genome organization

• When the retrovirus genome enters

a cell, the RNA gets translated to

form reverse transcriptase, which

copies the viral RNA into DNA.

• This DNA then integrates into the

genome: it becomes a provirus.

• The provirus DNA is transcribed to

make more viral RNAs and

proteins.

• The virus buds out through the cell

membrane and then infect another

cell.

• The integrated provirus has three

genes (gag, pol,and env).

gag gene encodes a

viral structural

protein

pol encodes the

reverse transcriptase

and integrase

Genome organization

• The terminal regions of the RNA

genome are duplicated in the DNA

as long terminal repeats (LTRs).

• U5: A unique, non-coding region of

75-250nt which is the first part of

the genome to be reverse

transcribed, forming the 3' end of

the provirus genome

• U3: A unique non-coding region of

200-1,200nt which forms the 5' end

of the provirus after reverse

transcription; contains the promoter

elements responsible for

transcription of the provirus.

Retroviruses as vectors

• The retroviruses are modified to carry genes.

• The gag, pol, env genes are deleted rendering them incapable of

replication inside the host cell.

• Viruses are then introduced into a culture containing the helper

viruses.

• The helper virus is an engineered virus which is deficient in Ψ

segment, but contains all other genes for replication. That means

it has the genes to produce viral particles but lacks the genes

required for packing.

Gene therapy with Retrovirus

• Replication defective vectors are the most common choice in

studies because the viruses have had the coding regions for the

genes necessary for additional rounds of virion replication and

packaging replaced with other genes, or deleted.

• These virus are capable of infecting their target cells and delivering

their viral payload, but then fail to continue the typical lytic

pathway that leads to cell lysis and death.

Retroviral vectors can either be replication-competent or

replication-defective.

Gene therapy with Retrovirus

• Replication competent viral vectors contain all necessary genes for

virion synthesis, and continue to propagate themselves once infection

occurs.

• Because the viral genome for these vectors is much lengthier, the

length of the actual inserted gene of interest is limited compared to

the possible length of the insert for replication defective vectors.

• Depending on the viral vector, the typical maximum length of an

allowable DNA insert in a replication defective viral vector is usually

about 8–10 kB.

Retroviruses as vectors

• The replication deficient but infective retro virus vector carrying the human gene now comes out of the cultured cells.

• These are introduced in to the patient. The virus enters the cell via specific receptors.

• In the cytoplasm of the human cells, the reverse transcriptase carried by the vector converts the RNA in to DNA, which is then integrated in to the host DNA.

• The normal human gene can now be expressed. The integrated DNA becomes a permanent part of the chromosome.

Drawback using retroviruses

• The recombinant retroviruses such as the Moloney murine

leukemia virus have the ability to integrate into the host genome

and involves the requirement for cells to be actively dividing for

transduction.

• As a result, cells such as neurons are very resistant to infection

and transduction by retroviruses.

• There is concern that insertional mutagenesis due to integration

into the host genome might lead to cancer or leukemia.

• This concern remained theoretical until gene therapy for ten

SCIDX1 patients using Moloney murine leukemia virus resulted in

two cases of leukemia caused by activation of the LMO2 oncogene

due to nearby integration of the vector. (FDA approved )

Lentivirus and their role in Gene Therapy

• They have recently been adapted as gene delivery vehicles (vectors)

because of their ability to integrate into the genome of both dividing

and non-dividing cells.

• Viruses in this genus include human immunodeficiency virus (HIV),

simian immunodeficiency virus (SIV), and feline immunodeficiency

virus (FIV).

• Their exogenous characteristics and successful gene integration

properties make them incredibly ideal for use in gene therapy.

Introduction to Lentivirus (Complex Retrovirus)

Retroviridae

Orthoretrovirinae

Alpharetrovirus

Betaretrovirus

Deltaretrovirus

Epsilonretrovirus

Gammaretrovirus

Lentivirus

Spumaretrovirinae Spumavius

Family Sub-Family Genus

Genome organization

• Infectious viruses have three main genes, 5´-gag-pol-env-3´.

• Two regulatory genes :

• Tat- It is an RNA binding protein that enhances transcription. and

• Rev- It is an RNA binding protein that promotes late phase gene expression. It is also important for the transport of the unspliced or singly spliced mRNAs, which encode viral structural proteins, from the nucleus to the cytoplasm..

• There are additional accessory genes depending on the virus (e.g., for HIV1: vif, vpr, vpu, nef)

• Nef protein inhibits T-cell activation.

• Vpu enhances the release of the virus from the cell surface to the cytoplasm during entry.

• Vpr is required for virus replication in non-dividing cells

• Vif protein is necessary for replication of lentiviruses due to its ability to down regulate the host’s antiviral response.

Characteristics

• 1. Lentiviruses have the ability to integrate into the genome of dividing and non dividing cells.

• 2. Lentivirus genomes encode regulatory proteins.

• 3. Lentiviruses persist lifelong.

• 4. Lentiviruses have high mutation rates.

• 5. Infection proceeds through at least three stages.

• (A) Initial (acute) lentivirus infection is associated with rapid viral replication and dissemination, which is often accompanied by a transient period of disease.

• (B) This is followed by a latent period, during which the virus is brought under immune control and no disease occurs.

• (C) High levels of viral replication then resume at some later time, resulting in disease.

Lentiviral Vectors

• Lentiviral vectors can deliver large cDNA’s to a variety of dividing and

non-dividing cells, including terminally differentiated mammalian

cells, e.g. neurons, lymphocytes and macrophages.

• Effective in transducing brain, liver, muscle and retina in vivo without

toxicity and immune responses.

• In gene therapy of diseases like :

AIDS

Diabetes mellitus

Murine hemophilia A

Prostate cancer

Chronic granulomatous disease and

Vascular gene therapy.

Making Lentiviral Vectors

Here the packaging cells produce infectious particles, whose genome only

encodes sequences from the transfer plasmid, which can be used to transduce

the target cells.

• This vector is used to transfer

genes of interest into the

target cells.

• There is a deletion of the U3 region

and other transcriptionally active

sequences from the 3’ LTR, which

results in it being a self inactivating

LTR.

• The 5’ LTR drives expression of the

packaged genomic RNA, and the

transgene is driven from a promoter

within the vector.

Contd..

• HIV particles are allowed to infect the transfected cells, where they package and thus carry only the gene of interest.

• This complex process is achieved through the design and organization of at least three transfected plasmids.

• The current Lentiviral vectors used are third generation Lentiviral vectors, which have been corrected to ensure safety and prevent the formation of replication competent Lentiviuses (RCLs) through recombination between plasmids.

• Together, the plasmids contain only the three essential retroviral genes plus the transgene separated across the three distinct transfected plasmids.

• By deleting unnecessary proteins and allocating parts of the genome to different plasmids, it is extremely unlikely to acquire replication-competent lentiviruses.

The three generations of Lentiviral vector genomes

• Plasmid 1 contains a promoter and viral

genes gag and pol provided in trans,

both necessary to generate a functional

virus.

• Plasmid 2 contains the transgene acting

in cis inserted into the original

lentiviral genome after virulence genes

vif, vpr, vpu, rev, tat, and nef have been

removed from the original plasmid.

The transgene is located behind the

packaging signal (Ψ).

• Plasmid 3 contains the third essential

gene, env, encoding for the envelope

glycoprotein, as well as a receptor

binding domain (SU) that can be

altered to increase cellular tropism.

Using Lentiviral Vectors to fight HIV:

• HIV is a very effective Lentiviral vector because it has evolved to

infect and express its genes in human helper T cells and other

macrophages.

• Lentiviruses can be used to target key virus or host genes to confer

resistance to the HIV1 virus.

• This can be achieved by either targeting essential viral genes, such as

replication machinery, or targeting host genes that are involved in

allowing viral entry into the cell.

• HIV1 virus can thus be used as a gene therapy vector through the

replacement of virulence genes with therapeutic transgenes.

Development of HIV1 as a Gene Therapy Agent

• These modified viruses are allowed to infect cells, where they

undergo their natural infection cycle resulting in the insertion of the

therapeutic genes instead of proviral genes.

• One promising example of using HIV-1 to fight HIV involves a vector

that contains three anti-HIV transgenes.

• Anderson et al (2009) have developed a vector system that targets

both host and viral genes to successfully confer HIV resistance.

• They have shown that transforming CD4 + hematopoietic progenitor

cells with a CCR5 short hairpin RNA gene, a TRIM5α gene, and a

transactivation response element gene, each which operate at different

stages of the viral life cycle, successfully block HIV infection.

Application of Lentiviral Vectors in HIV-Targeted

Gene Therapy

• The Lentiviral vector created contained three anti-HIV therapeutic agents that function to interfere with infection at different stages of the replication cycle.

• The transgene that acts first in the replication cycle is the CCR5 short hairpin RNA (shRNA) that acts to prevent entry of the virus into the host cell.

• The CCR5 gene was proposed as a target for HIV gene therapy after the discovery that people homozygous and heterozygous for a CCR5 Δ32 mutation are naturally immune to HIV, and individuals lacking any CCR5 gene copies are functionally normal.

• When an HIV patient was given transplants of T-cells homozygous for the Δ32 mutation his HIV reached undetectable amounts.

• Furthermore, the CCR5 gene is a good candidate for gene therapy because it is a host gene, and thus will not undergo quick mutation and resistance selection like viral genes.

• The CCR5 shRNA in the vector construct acts similarly to miRNA interference, resulting in the silencing of the CCR5 gene.

Contd.. Application of Lentiviral Vectors in HIV-

Targeted Gene Therapy..

• The second transgene included in the vector was a chimeric human

and macaque version of TRIM5α to prevent integration of the provirus.

• Certain monkey TRIM5α isoforms prevent HIV infection by

interfering with the opening of the virus protein capsid after entry.

• The DNA encoding the monkey amino acids responsible for conferring

HIV resistance were fused to human TRIM5α gene in order to prevent

the protein from being rejected by the immune system.

Contd.. Application of Lentiviral Vectors in HIV-

Targeted Gene Therapy

• The third transgene mimics the viral transactivation response (TAR)

element and is controlled by the virulence tat gene to prevent

transcription of the viral genome.

• Functional TAR elements are recruited by the tat gene to bind to

elongation factors during transcription in order to catalyze the addition

of nucleotides to the growing chain by polymerase enzymes.

• The decoy TAR element used still binds to tat during transcription, but

is modified to prevent transcription rather than participate in it.

Contd.. Application of Lentiviral Vectors in HIV-

Targeted Gene Therapy

• The Lentiviral agent containing the TAR decoy, TRIM5α, and CCR5 shRNA were introduced into HPSC cells and their expression was confirmed through quantitative real-time PCR (QRT-PCR), with transgene expression being 30 to 4,000 times higher than control cells.

• In a later trial, when this Lentiviral vector was used to introduce the three-gene construct in order to confer HIV resistance into a mouse model with a humanized immune system, the group successfully generated an immune system capable of resisting HIV infection.

• While not yet available for human use, the construct designed by Anderson et al (2009) represents just one of the significant achievements in HIV-1 derived vector gene therapy to cure HIV positive individuals.

Timothy Ray Brown

• He was diagnosed with the disease in 1995. After using

antiretroviral drugs to stave off HIV for more than a decade,

he received another staggering diagnosis: acute myeloid

leukemia.

• Brown’s doctors decided to use radiation and chemotherapy

to wipe out his immune system, then rebuild it with donated

stem cells.

• But they added a game-changing twist to this standard

treatment: They deliberately picked a donor who was

immune to HIV (cells don’t have CCR5, a protein that opens

the door for HIV to enter blood cells.)

• Brown received two stem cell transplants that knocked out

his cancer and transferred the genetic variation to his

immune system curing both leukemia and HIV..

Timothy Ray Brown,

also known as the

"Berlin patient," was

cured of HIV in 2008.