Vectors Modes Of Gene Transfer Biology Essay

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Foetal gene therapy is one of the promising novel scientific strategies to correct genetic disorders. In current scenario, it has been specifically considered to be an auspicious strategy to overcome the limitation of conventional haematopoietic stem cells (HSC) therapy by evading the allogenic HLA barriers, using genetically corrected autologous HSC in foetus.

Vectors (Modes of Gene Transfer)

Generally vectors used for the gene delivery to the cells bind specifically to the targeted organ and require just a single application. Though the non viral vectors such as gene gun have been proposed to be safer mode of transduction, but the transgene introduced by them remains episomal or lost with cell division, and thus renders limited expression. (iii) Hence viral vectors are considered to be more efficient vehicles for gene delivery due to their ability to penetrate host cells and replicate inside the host cell. Before transfection, they are engineered to attenuate their genome so that only the transgene , not the viral genes are copied. There are several factors like immunogenicity, packing capacity, targeted tissue and desired duration of expression that play vital role on the choice of a viral vectors.

(see the book )

Past experiments show that Adenoviruses have been used in some of the models of foetal gene transfer. But they were not found to integrate into host genome. As a result transgene expression was found to be instantaneous in rapidly dividing foetal cells. Moreover, their high imunognecity prevented them to be a good vector for in utero gene therapy. Adeno associated viruses(AAV) are comparatively less immunogenic and their serotype influences tissue specificity. But due to their tendency to integrate into the genome at low frequency and their slow expression profile, it may take few weeks for AAV to reach to peak expression level. Relatively longer duration of transgene expression can be seen in the tissues like central nervous system, liver and skeletal muscles, where there occurs less turnover of cells. Similarly the vector must integrate into the host genome in order to ensure long term expression of the transgene.

Ideally foetal gene therapy requires a vector system of specific gene construct that reach to the required organs and leave its permanent expression avoiding the germ line transmission. Unfortunately no single vector is available to meet this goal. However due to their capacity to render long term expression of gene, viral vectors like adeno viruses, retro virus and adeno associated viruses are relatively more suitable options for foetal gene therapy. (Gregoriadis G., Mc Cormack B., 2000).



3.1 Route and Timing

After selection the suitable vector, transduction of the targeted population of stem cells is carried out, which depends on site of vector administraton and gestational age of the foetus. Comparatively, earlier gestational gene therapy produce better results of transduction of stem cells than the later gestation gene therapy, because stem cells applied in earlier gestational life will integrate into tissue and differentiate along with the growth of the foetus.

Transuterine injection guided with ultrasound is the most common way of prenatal gene therapy in animal models. Directadministration into the lung, liver, and brain has led to localized

gene expression,31-33 whereas vector injected into a body cavity

such as the peritoneal or amniotic cavities can potentially transduce

several different progenitor cell populations. Developmental stage

at the time of injection will determine which progenitor cell populations

are exposed to vector and more than one population may

be transduced at a time.(3)(Roybal et al.)

Ex vivo approach

Major concern of ex vivo approach of foetal gene therapy is to target autologous haematopoietic stem cells in vitro, enabling them to be highly proliferative and able to give enhanced gene expression. In this approach, the therapeutic gene is transduced to the autologous HSC in vitro and transplanted back to the foetus. GIVE EXAMPLES LIKE SCID ETC



In vivo approach

In vivo approach of foetal gene therapy involves the direct transfer of vector gene construct into the foetus, leading to in vivo transduction of foetal HSC. This technique has the threat of germ line transduction of the gene. (Wngner,A.M.,Schoeberlein,A.,Surbek,D, 2009)

Candidate diseases

Being an experimental concept, foetal gene therapy can currently be targeted for future application in the life threatening monogenic diseases which are caused by absence or inactivation of essential gene product and which manifest themselves in foetal life. Similarly, having tendency to cause irreversible damage to organs in prenatal or early post natal life, is another criteria for the candidate diseases of the foetal gene therapy. Genetic disorders like cystic fibrosis, Phenylketonurea, Haemophilia, Epidermolysis Bullosa ,DMD, ADA deficiency and different haemoglobinopathies are the potential candidate diseases for foetal gene therapy. (Gregoriadis G., Mc Cormack B., 2000).

Cystic fibrosis is one of common autosomal recessive monogenic disorder having incidence of 1 in 2000 in new borns. (ADM Jacksin, 1989 the natural history of cystic fibrosis, in : cystic fibrosis (ed P Goodfellow) Oxford University Press 1-11). The genetic cause behind the onset of this disease is the mutation of gene coding Cystic Fibrosis Transmembrane Regulator (CFTR) protein. This disease mainly affects lungs, pancreas biliary tract and intestine, causing shortened expectancy. Appearance of CFTR protein in human embryo occurs by 7 weeks of gestation in the yolk sac and shortly afterwards in ciliated tracheal cells. This protein is expressed in intestine at 12th week , and apical domain of ciliated airway epithelia in 24th to 25th week of gestation. (Ref 5/book) Thus it shows its vital role during the development of human foetus. Hence gene therapy in case of cystic fibrosis is most relevant during prenatal rather than post natal life.

Another lung disease caused by deficiency of surfactant protein B affects the expansion of neonatal lung after birth. This is another candidate disease for foetal gene therapy and can be treated by delivering the gene encoding this protein , in utero. Similarly, metabolic disorders (e.g. phenylketonuria ), or storage diseases that occur during prenatal life are other pathological conditions that manifest themselves in foetal life and ideal target for gene therapy in utereo.

In the scenario of expensive treatment and problems associated with repeated transfusions, haemophilia is one of the best example of candidate disease for the application of foetal gene therapy. Due to the immune responses, post natal gene therapy does not seem to prevent manifestation of haemophilia (41/ journal 3). The other factors that make haemophilia an ideal candidate disease for foetal gene therapy are: availability of its prenatal diagnosis, etiology being loss of function of a single gene, and its known pattern of inheritance.

Haemophilia has been studied extensively in animal models with the transfer of required gene at various gestational age through different routes using different vectors. Schneider et al had compared intraperitoneal, intramuscular, and intravenous injections of human factor IX via adeno virus and AAV-2, in E 15.5 foetuses of mice . As a result , initial higher level of factor IX was seen in foetuses transfected with adeno viral vectors. It was followed by decrease in the expression of the transgene in both vectors over time However, adeno viral vector injected mice maintained the therapeutic level for sx months. But no antibodies were formed against either vector or transgene. (42/3)Sabatino et al. observed low-level human factor IX expression after intramuscular injection of AAV-1 and -2 in E14 fetuses and neonatal mice. Tolerance induced to AAV-1 but not AAV-2 allowed the postnatal re-administration of the AAV-1-driven transgene and

subsequent therapeutic factor IX levels.43/3 The most impressive results of prenatal gene transfer in hemophiliac mice occurred with a lentiviral vector. Waddington et al. demonstrated therapeutic levels of factor IX expression (9-16%) for 14 months, improved coagulation, and no immune response against the protein following intravenous administration of a lentiviral-driven transgene into E15 fetuses.44 These studies have shown that prenatal gene therapy in mice can result in low-level transgene expression that not only has therapeutic significance, but also in some cases induces tolerance allowing postnatal administration of the same vector and transgene for enhancement of expression.

Experiments on Animal Models and Route of Administration

Foetal gene therapy has been performed in mice, rats, rabbits and sheep so far. Out of them foetal sheep are the most ideal models of foetal gene therapy because of their physiological and developmental similarities with humen. Using different rodent models for human genetic diseases and various routes of administration, foetal gene therapy has been aimed to target different organs. However, the models for pre clinical foetal gene therapy are still under investigation. Following routes of administration can be performed on different animal models:

Intra amniotic delivery

Systemic delivery through foetal circulation

Gene delivery to extra pulmonary tissues in post implantation embryonic experimental animals: (Gene therapy protocols)

Most of the current preclinical approaches relate foetal gene therapy with the transduction of foetal heamopoietic tissue. Many laboratories have been successful on to transfect bone marrow cells in culture. But Problems like longevity of expression, difficulty on obtaining and transducing totipotant stem cells and potential requirement of bone marrow ablation have prompted alternative strategies.

Gene transfer to foetal lung:

Gene therapy to mature lung have undergone through [lenty of clinical trials for the potential therapy against cystic fibrosis. Respiratory epithelium is the most favourable site for the application of gene therapy. Following are some of the points that rationalize the application of adenoviral mediated gene theapy on foetal lung:

Limited immune reposnse and minimized inflammatory response on the foetus, prolonging the adenovirus mediated transgen expression (22/new book)

Accessibility to plenty of dividing cells (may be stem cells) that allow the use of retro viral vectors.(23/new book)

More expression of CFTR on foetus than adult lungs (24, 25/ new book )

Less complex surface lining of lungs and presence of limited number of macrophages and proteins that facilitate the delivery of DNA .

Following is the description of adeno-virus mediated gene transfer protocol in foetal lung. It was carried out by Pitt, Schwarz and Bland in sheep model.

Material and Methods:

Production of Retroviral supernant :

MFG retro viral vectors inserted with LacZ or secreted human interleukin receptor antagonist protein cDNA were used . This MGF vector is the simplified retroviral vector derived from M-MuLV devoiding the polumerase and envelope gene sequences. However partial gag sequences were tetained to increase the packing efficiency of the unspliced transcript. The inserted cDNA were transcribed from the promoter/enhancer sequences from the retroviral long terminal repeat(LTR).High titer amphotrophic producer of recombinant retroviruses were produced by cotransfection of plasmid (pSV2neo) into the packaging cells(CRIP). NIH3T3 cells were cultivated in DMEM and provided with 4.5 g/L of glucose and 10% (v/v) heat -inactivated sheep serum, 100 U/ml of penicillin, 200 mg/mL of glutamine, and finally infected with virus. The medium containing viral supernatant was tested for the viral titre and presence of helper virus , concentrated 3X and stored at -70degree celcius.

Foetal Surgery:

Twin foetal lambs were surgically prepared with catheters and the ewe was sedated intramuscularly with 15 mg/kg ketamine. After that hysterotomy was performed with halothane and nitrous oxide anaesthesia. The uterus was, then, opened with a small incision and polyvinyl catheters were placed directly into the foetal carotid artery, jugular vein and trachea. Similarly, a fluid- filled catheter was placed in the amniotic sac, and the catheters were sutured to uterus and abdomen of ewe. The wounds were closed with silk sutures , and catheters were placed in a pouch sewn on the flank of the ewe. Amniotic sac was administered with 1000,000 U of penicillin , and 400 mg of kanamycin during and after surgery. Similarly,foetal vein was administered with 300,000 U of penicillin and 10 mg of gentamycin at the time surgery and daily after that. The ewe was given 5 mL mixture of penicillin and dihydrostreptomycin and 600 mg of kanamycin intramuscularly each day.

Administration of Vector to the Foetus:

After 1 day of surgery, blood was withdrawn from the carotid artery catheter for the determination of haematocrit, pH and blood gas tension. 60 mL of tracheal fluid was withdrawn into a sterile syringe. After this, 10 mL solution of MFG-viral supernatant having 3x 10 6 particle per mL concentration was brought to room temperature immediately before use and polybrene was added to a final concentration of 2 micro gram per mL. The viral supernatant was delivered directly to the tracheal catheter and flushed to the lungs with 30 to 60 mL of previously withdrawn lung liquid. Same procedure was carried out for other twin with other MFG-vector. All the catheters were flushed with heparinised saline and antibiotics was given as per the pre defined dose. This whole procedure was repeated for three consecutive days.

Histochemical Techniques:

The ewe and the foetuses were killed with an intravenous overdose of sodium pentobarbital (50 mg/kg).Their lungs were removed and inflated ex vivo to 30 cm H20 transpulmonary pressure by injecting phosphate buffered solution of pH 7.4 (containing 4% paraformaldehyde and 0.1% gluteraldehyde). After that lungs were immersed into phosphate buffered saline containing 30% sucrose and incubated at 4 degree Celsius.

For histochemical analysis, small blocks of tissue were obtained from proximal and distal section of each lobe and frozen in liquid nitrogen. Frozen sections were cut with cryostat and mounted on poly-L-lysine -coated glass slides.

To localize beta galactosidase activity, small blocks of fixed tissues were rinsed in PBS and incubated in a solution containing 1 mg/mL of 5 bromo 4 chloro 3 indoyl- D-galactopyanoside in Tris-phosphate buffered saline (pH 8.0) in 5mM potassium ferricyanide and potassium ferrocyanide. The tissue sections were further incubated for 8 hours and washed in PBS. After that tissue was frozen and sectioned, followed by counter stain with eosin and haematoxylin. The stained sections were observed under microscope. Similarly, immunohistochemistry was performed on these samples.


Detectable levels of beta galactosidase was noted in the upper and lower airways of some of these animals, for 3 weeks after infection. Localization of beta galactosidase was found to be the most prominent in the epithelial cells of proximal airways with additional histochemical appearance associated with fibroblasts and macrophages in the sub mucosa.

This experiment was performed using MFG-based replication-defective retroviral vectors to express beta galactosidase and human interleukin receptor antagonist protein in the lungs of foetal sheep (book 2/37). This approach is most applicable to inherited disoreders like cystic fibrosis or surfactant apoprotein B deficiency where potential life time therapy may be exacted after limited therapeutic application in utero.

fetuses and have large fetuses that are amenable to early gestational

manipulation. In addition, the ovine immune system has

been extensively studied and the ovine life span is long enough to

allow for long-term studies of safety and efficacy. Sheep studies

have compared different injection techniques and vector types, but

only low-level expression has been reported. For example, injection

of retroviral vectors into preimmune fetal sheep was shown to

transduce hematopoetic cells with low-level expression observed

for 5 years.38 Intra-tracheal adenoviral and lentiviral injections

have led to low-level gene expression in peripheral airways.39,40

Further research with large animal models is needed to refine the

technical aspects as well as the timing of potential fetal gene

therapy in humans results of prenatal gene transfer in hemophiliac mice occurred with

a lentiviral vector. Waddington et al. demonstrated therapeutic

levels of factor IX expression (9-16%) for 14 months, improved

coagulation, and no immune response against the protein following

intravenous administration of a lentiviral-driven transgene into E15

fetuses.44 These studies have shown that prenatal gene therapy in

mice can result in low-level transgene expression that not only has

therapeutic significance, but also in some cases induces tolerance

allowing postnatal administration of the same vector and transgene

for enhancement of expression (3)

Table : Significant endeavours in the field of foetal gene therapy in animal models in metabolic, central nervous system and musculo-skeletal diseases [Adopted from :




Animal model







Cystic Fibrosis





Reversal of the fatal cystic fibrosis

Larson et al.



Crigler- Najjar



Lenti virus


Decreased serum bilirubin by 45% for >1 yr ,but developed antibodies




Pompe's Disease




Intrahepatic, intraperitoneal

Transduction of the diaphragm with normal contractile function for 6 months

Rucker et al.



Leber's congenital amarosis

RPE 65




Restoration of vision for 2.5 months, gene expression for 6 months

Dejneka et al.



MPS VII (Sly syndrome)

Beta Glucuronidase




CNS expression for 1 yr

Karolewski et al



Duchenne's muscular dystrophy





Gene expression in 2-3 muscles of injected hindlimb, muscle degeneration at 9 weeks

Reay et al


(CFTR : Cystic fibrosis transmembrane conductance regulator, UGT 1A1: UDP-glucuronyl transferase, AAV : Adeno associated virus, RPPE 65: Retinal pigment epithelium 65, MPS VII: Mucopolysaccharidosis VII)

Foetal gene therapy has been performed in mice, rats, rabbits and sheep so far. Following routes of administration can be performed on different animal models:

Intra amniotic delivery

Systemic delivery through foetal circulation


Most of diseases presently treated by cell transplants

in the perinatal period are genetically-determined,

inherited disorders. It is tempting to

propose gene therapy either postnatally or even

prenatally to these patients.

SCID patients with ADA deficiency have been

treated, ten years ago, with ADA gene therapy ex

vivo. Using a retroviral vector, the ADA gene has

been transferred to T lymphocytes from the patients

and the corrected cells were infused back to

the patients, with a demonstrable but partial

benefit [21]. More recently, the X-linked SCID

has been treated by gene therapy into precursor

CD34_ lymphocytes of the bone marrow, with a

more complete effect [22].

Experimentally, we have observed a larger efficacy

of gene transfer into fetal liver than into

bone marrow stem cells. Perspectives of in utero

or neonatal gene therapy are the following:

ex vivo transfer of the appropriate gene and

vector into fetal cells then in vivo administration

of the cells (in utero);

in vivo direct injection of the appropriate gene

and vector (in utero);

ex vivo transfer of the gene and vector in cord

blood cells at birth, then administration to the


Special care should be devoted to safety and to

prevention of gene transfer to germ-line cells.(7)

Commercial aspects

Contemporary Scenario

Genteric Inc., Gene Medicine Inc., Gen Vec Inc., etc are some of the pioneering biotechnological companies involved in business of gene therapy. They have been focussed on providing new generation gene therapy and hold the patents of different biotechnological productions.

Future Implications

Due to some of its merits over the post natal gene therapy, foetal gene therapy shows a lot of possibilities to emerge as a promising field of biotechnology market.

Ethical aspects

Safety and potential risk

Potential risk of germ line transduction of theraupetic gene in foetal therapy raises a large number of social, legal and moral questions. Similarly the possibility of insertional mutagenesis in foetal cells leading to gene defect or formation of malignant tumour is another aspect of foetal gene therapy that creates ethical problem.( Noble,R., Rodeck C.H., 2008).

At the present time, there are

well-documented risks associated with viral vector-mediated

gene transfer that need to be addressed prior to consideration of

any clinical application in the fetus. However, if safe and effective

methods for fetal gene transfer can be developed, a large number

of disorders would be amenable to IUGT. There is an increasing

body of experimental evidence that supports the therapeutic

potential of IUGT.(3)

The potential safety concerns of prenatal gene therapy include

those associated with fetal intervention and those due to gene

transfer. As with any fetal intervention, fetal loss, infection, and

preterm labor are possible. In reality, a minimally invasive approach

using a fine needle under ultrasound guidance has minimal procedure

related morbidity. As previously discussed, some gene transferrelated

risks depend on the type of vector. The host immune response

to vector or transgene, insertionalmutagenesis caused by integrating

vectors, and the risk of a replication-incompetent HIV vectors

becoming replication-competent may all be concerns depending

upon the gestational age of the recipient and the vector construct

used. Common to all vectors used for prenatal gene transfer are the

concerns of germ-line transmission, disruption of normal organ

development, and transplacental spread of transgenes to themother.

Lentiviral vectors result in efficient transduction because they

integrate into the host genome, but the DNA insertion site may have

deleterious consequences. New mutations have been observed

after postnatal gene therapy with integrating vectors. Four cases of

T-cell leukemia were diagnosed 31-68 months after retroviralmediated

gene therapy for X-linked SCID.45 Only one prenatal study

has demonstrated the same phenomenon. A high incidence of liver

tumors was observed in mice that received prenatal injection with

an early form of third-generation equine infectious anemia virus

vectors with self-inactivating configuration. The insertion sites

were not identified, but no tumors were observed when a similar

vector with an HIV backbone was used.46 Further studies of

prenatally treated animals are needed to fully assess the risk of

insertional mutagenesis.

Although prenatal gene transfer has great potential for restoring

normal function, manipulation of the fetus may alter normal organ

development. Both the site of injection and the toxicity of the

vector itself need to be evaluated. Sheep subjected to in-utero

intrapulmonary and intracardiac vector injection showed no

adverse effects on the postnatal heart and lung development.47

However, we have found that fibroblast growth factor 10 expression

in the developing rat lung leads to cystic adenomatoid malformations

illustrating how forced expression of a specific

transgene can lead to malformation.48

Prenatal gene therapy is directed toward somatic cells, but

inadvertent gene transfer to the germ line is a major concern for

both safety and ethical reasons. Targeted gene therapy that occurs

after the compartmentalization of primordial germ cells should not

affect the germ line.49 Gene transfer to the germ line has been

investigated by several groups. Porada et al. evaluated sheep that

received intraperitoneal retroviral-mediated prenatal gene transfer.

Despite negative breeding studies, PCR on the purified sperm

from injected rams and immunohistochemistry of sectioned testes

showed low-level transduction of germ cells.50 More recently, the

same group reported that gestational age affects germ cell transduction.

Given the likelihood that low-level transduction of germ

cells after systemic administration of integrating vector to the fetus

cannot be completely excluded, the frequency of germ-line transduction that is acceptable in the context of treatment of

a severe genetic disorder needs to be considered (3)

Foetus as a Patient: Conflict of duty

In foetal gene therapy, there is probability of conflict between the perceived duties and obligation of clinician and those of pregnant woman. Similarly the potential conflict between duties owed by clinician to the pregnant women and perceived obligation to the well being of the foetus might create ethical problems. Such situation might arise when the pregnant woman refuses a therapeutic procedure that has been recommended by the clinician for the well being of the foetus. ( Noble,R., Rodeck C.H., 2008)

Informed Consent and Human Trials

Informed consent is a basic concept of medical research involving human participants where potential harms and benefits of the research are thoroughly informed to the participant and they are considered to be the beneficiaries of the research. But in foetal gene therapy, pregnant woman who is considered to be the participant of the therapy is not the direct beneficiary of the therapy, but her future child. Since these participants might have to carry a burden of risk, it might be unethical to recruit them for a therapy that might benefit their offspring, until we are uncertain about the success of this therapy. ( Noble,R., Rodeck C.H., 2008)


Fig Approval process of human gene therapy (Sources: Nichols 1988, Human Gene therapy, National academy of sciences, page 168


Despite its some of merits over the post natal gene therapy, foetal gene therapy is still under experiment and bears lots of potential threats and ethical issues. This technique must undergo a series of trials in suitable animal models to prove its safety and efficacy before initiating clinical trials on human beings. Fetal gene therapy has even greater potential to prevent the

onset of inherited genetic diseases, but it is still in the early

experimental stage. Proof-in-principle for fetal gene therapy for

many disorders has already been demonstrated in rodent and large

animal models. Safety concerns involving the risk of insertional

mutagenesis, the effect on organ development and the importance

of low-level germ cell transmission need to be extensively investigated

in appropriate preclinical animal models prior to application

in humans. The ethics of fetal gene therapy and its potential to

alter the human genome also need to be considered. While greater

tissue specificity and safety can likely be accomplished by the use of

tissue-specific promoters, or regulated transgene expression, safer

gene transfer technologies will need to be developed to alleviate

these concerns. (3)

Despite the recent advances in foetal therapy, we are still in need

of a strong evidence of efficacy and safety both to the mother and

foetus in many of the exciting therapeutic approaches, possibly

provided by the results of ongoing randomised trials.(4)