Gene Transfer Vectors for Clinical Application: 507 (Methods in Enzymology)

Recent trends in the gene therapy of β-thalassemia
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The target cell niche in this study was the expanding population of fetal liver haematopoietic stem cells. There are intrinsic differences between fetal and adult HSCs properties especially fetal liver HSCs which can give rise to thymocytes, especially Tregs which are responsible for promoting tolerance to self and potentially foreign antigen The fetal to adult switch in these HSC properties occurs between week one and two after birth in mice. During fetal life, the HSC pools in the fetal liver are actively cycling and rapidly expanding whereas, once the HSCs reach the fetal bone marrow, they become quiescent.

This property of fetal HSC pools in the liver and their potential of thymic repopulation may be an added advantage for intrahepatic IUGT 48 which is easily able to direct gene transfer to the fetal liver through direct injection. Fetal liver HSCs are also capable of repopulating five times more effectively long term than short term Any manipulation of liver HSCs should have a long-term effect, ideally permanent, especially if these cells migrate to the thymus Intrahepatic fetal gene transfer to haematopoietic progenitors has also been demonstrated in primates where the maximum effect was achieved using lentiviral vectors Correction of haemoglobin was achieved with a low VCN of 0.

This is similar to the findings of Miccio et al. This can be explained by the fact, that possibly, a large percentage of non-hematopoietic off-target cells were transduced following the direct intrahepatic lentiviral injection.

Other authors have demonstrated that non-haematopoietic cells are transduced in an intrahepatic IUGT. The developmental stage of each organ, in this case the liver, could determine the susceptibility to in utero gene transfer and transgene expression The efficiency and distribution of transduction, is also highly depended on the type of vector used, as well as, the route of administration This could raise safety concerns, even though in a Cynomolgus Macaque model no tumours were observed, 6 years post IUGT, with an Adeno Associated viral vector.

The authors, in this non-human primate study, addressed the importance for life long surveillance The use of direct in utero gene therapy, took advantage of the high proliferation rate of the fetal HSCs which resulted in an adequate number of transduced daughter cells that were able to correct the phenotypic Hb defect. It was not possible to do individual sorting of HSCs from each transduced mouse due to the minimal amount of tissue collected and the scope of analysis initially planned such as EMH, histology and HPLC. Thus, we were unable to calculate the VCN in the HSC population only and instead present data on the VCN for the whole heterogeneous population of hepatocytes and whole bone marrow.

It is possible that the HSCs might have had a greater percentage of initial correction since the fetal liver is mainly made of HSC progenitors. Alternatively this result may not be true as we concentrated on analysis of the whole bone marrow rather than just the BM HSCs and our VCN result may have been lowered due to dilution.

Further studies are needed to confirm this effect.

Vectors of recombinant genes

New England Journal of Medicine, , Clonal abundance is calculated as the sequencing reads representing each integration site within each data set. For example, to prevent thrombosis in nondenuded vessels, endothelial cells are the target. Circ Res. So, transposon-based gene delivery systems such as Sleeping Beauty [78,81,83] or PiggyBac [71,79,80] allow stable gene transfer into T cells and an efficient expression of the therapeutic transgene. Korner A, Pawelek J.

Previous studies have found low genotoxicity of lentiviral vector integration in mice that are prone to developing tumours Nevertheless, it is an important safety consideration to keep the VCN as low as possible whilst maintaining the efficacy of the gene transfer. The humanised mouse model of beta thalassaemia 14 , 15 , 17 , 19 that we used is unique in that the fetal mice produce human gamma-globin human fetal haemoglobin in utero 15 , and have a human gamma to beta switching cassette which results in the death of homozygous animals within two weeks of birth, as the production of defective beta globin takes over.

Homozygous animals can be rescued postnatally using regular blood transfusions This model allows human beta-globin producing vectors to be tested in utero 12 , 13 , 54 , 55 , and since the in utero phenotype is less severe than of other thalassaemia mouse models, eg th3 , it allows the evaluation of IUGT to rescue the mouse model in utero and post-natally 45 , In the first cohort 12 Weeks , where the animals were analysed at 12 weeks after birth, we observed normalisation of the phenotype with functional correction of anaemia, excessive extramedullary haematopoiesis and the other abnormal haematological indices.

The presence of normal blood films in the treated animals compared to untreated was a confirmatory result. To confirm the presence of normal Hb we used real-time PCR and high-performance liquid chromatography to demonstrate beta-globin gene expression with downregulation of the gamma-globin gene as well as the additional peak from the beta globin chain in HPLC. These results excluded the hereditary persistence of fetal haemoglobin which could have contributed to the phenotypic correction of the overall haemoglobin.

In the long term week postnatal group, the phenotypic correction appeared to be less effective as in the group analysed at 12 weeks after birth suggesting that the effect may reduce over time.

Gene Transfer Vectors for Clinical Application, Volume 507

This could be due to transcriptional silencing of the transgene 57 or even due to epigenetic changes leading to deactivation of the beta-globin gene as the pups grew 58 , This was evident in the lower Hb concentration at the later time point 9. Histological analysis of the animals showed a correlation of spleen hyperplasia with iron deposits in the spleen, moderate anisocytosis and anaemia.

In the long-term group, MRI was used as a novel method of monitoring in utero gene therapy functional success left ventricular ejection fraction, spleen volume, iron overload 60 , 61 , 62 , It is also used for the assessment of the thalassemia major patient prior to stem cell transplantation This study confirms that this can be applied in small animal studies which can be used for the non-invasive assessment of new therapies All live cross-fostered pups survived to genotyping.

There are a number of possible reasons for this low survival of homozygote injected pups. It is possible that the treated homozygous pups demised in utero after the intrahepatic surgery or in the first hours after birth before they were cross-fostered due to a higher susceptibility to miscarriage or perinatal loss.

Pups were usually born overnight and immediate cross-fostering was not possible as there was not always staff available to transfer them to the foster mother. It is also possible that homozygote rescue was incomplete rendering the born homozygote newborn pups more susceptible to being rejected or attacked and eaten by their mother.

This has been reported in other studies where fetal intervention occurred Future studies using a higher titre of the vector will be conducted to determine whether this improves survival in the homozygous pups. In this study, we show that targeting the specific stem cell niche 66 , 67 early enough in gestation with gene therapy could be critical for fetal gene transfer, especially if most of the haematopoietic stem cells are within the compartment, in this case, the liver.

Alternative routes such as intraperitoneal and intravascular injection need investigation to achieve a phenotypic cure, especially rescuing the homozygous animals.


Boelig et al. Whether this achieves the same effect for fetal gene therapy remains to be seen. Importantly for clinical translation, the tissues were studied by an expert, and no animals were found to have malignancy, which is in keeping with other studies using this stable GLOBE vector 13 , The number of vector integration sites corresponded to the vector copy number, even though this was low. This could be a sign that if higher VCN is achieved, the level of haemoglobin could also rise. The integration site analysis revealed integration to be associated with the neural system, stem cells, spermatogenesis and also with an oncogene.

This must be carefully studied since the potential of tumor formation was shown to be high in a fetal context when using certain lentiviral vectors This also supports the use of an ex - vivo gene therapy approach using amniotic fluid stem cells or other highly efficient HSC progenitors One of the benefits of perinatal treatment lies in the potential to limit organ damage through early intervention. Also, applying a therapy to the fetus, where stem cell proliferation is high, results in a higher number of transduced cells, which leads to a better therapeutic effect The fetus also offers a size advantage, allowing a higher vector-to-target cell ratio.

Certain organs that are challenging to target after birth may be more easily accessible in the fetus due to their developmental stages or relative immaturity 72 , 73 , 74 , The fetal epidermis is an example of this, as it undergoes remodelling by programmed cell death and is replaced by mature keratinocytes. The epidermis forms a thick barrier to gene transfer following birth 76 but could be better targeted in utero One of the main obstacles of postnatal gene therapy is the development of an immune response against the transgenic protein or the vector This is of particular importance when gene therapy aims to correct a genetic disease, which completely lacks a gene product.

It is also possible that some patients may have pre-existing antibodies to the viral vector that will inhibit long-term expression of the transgenic protein; this will limit therapeutic efficacy and prevent repeated vector administration. For example, pre-existing neutralising antibodies against adeno-associated virus AAV serotype 2 have been shown to interfere with AAV2 vector-mediated factor IX FIX gene transfer to the liver 78 , 79 , 80 , Delivering foreign protein to the fetus can provide an advantage of immune tolerance during fetal life, a concept first suggested more than 60 years ago 82 , Induction of tolerance relies on the introduction and expression of the foreign protein early in gestation before the immune system is fully developed.

The protein needs to remain at a detectable level within the fetus and presented to the thymus at the correct time 84 , 85 , 86 , 87 , In a postnatal treatment this can be achieved by ex-vivo correction of autologous HSCs, which will prevent an immune response, since the immune system will recognise the autologous, corrected cells as self.

This can be demonstrated in a study, by A Thompson and colleagues. No complications or reactions to the vector were reported IUGT was delivered at embryonic day E Fetal gene transfer to the haematopoietic compartment 91 may offer a third option to couples with a prenatal diagnosis of a fetal congenital blood disorder, especially in the beta haemoglobinopathies For human therapy, non-invasive prenatal diagnostic techniques are being developed using maternal blood to diagnose fetal thalassaemia from 8—10 weeks of gestation This would allow enough time to deliver gene therapy to the human fetus using ultrasound-guided intrahepatic injection.

Using this methodology, no ex - vivo expansion and correction of the appropriate haematopoietic progenitors is required. More animal studies are required with the aim to rescue the homozygous mouse model. If this is successful, treatment could even be applied in low-income countries where the palliative cost of homozygous patients is unsustainable for the local health systems 94 , 95 , In order to reach the clinic, a gradual progression from small animal disease models, such as the mouse, followed by studies in larger animals, for example sheep to test the feasibility and safety of the delivery method will be necessary.

A reproductive toxicology study would then be required, which might be performed either in rabbits or possibly in non-human primates before establishing a fist-in-human clinical trial of fetal gene therapy 73 , Experimental protocols were performed following the U. Mice were housed in single cages after plugging. The dams were given wet food after the laparotomy and closely monitored for the first seven days after the procedure. IUGT was performed in timed-mated pregnant heterozygous thalassemia mice as described 38 , with minor modifications.

On E The uterus was returned to the maternal peritoneal cavity, the abdomen was closed using an absorbable vicryl suture Ethicon Inc. After spontaneous delivery at EE21 day of gestation, treated pups were cross-fostered to CD1 dams by day 2—3 after birth, to avoid maternal cannibalism and maternal antibody response to the transgenic protein , , Pups were genotyped at three weeks of age; earlier genotyping was precluded by the Home Office Project license.

The pups were bred until scheduled post-mortem examination at 12 and 32 weeks postnatal. At post-mortem examination, blood 0. The spleen and liver were placed in 1. For bone marrow collection, the femur and tibia were separated from the body and muscle was removed off the bones. The epiphysis was removed from both ends and placed in 1. The bone marrow was harvested by inserting a syringe needle Terumo, myjector 27G, 0.

The bone marrow aspirates were collected into 1. The reactions were done in 0.

Construction strategies for recombinant genes

Results were analysed using the Life Technologies Step-One software. Titin, also called connectin, is a protein kinase involved in the structural assembly of microfilaments in muscle and during chromosome segregation and cell division in non-muscle cells.

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The primers already described in the literature 19 were used for the study. Red blood cell counts and Hematocrit values were measured. Animal and Spleen weight was also measured using a high precision balance Kern, UK. The images were processed using Zeiss-lite software In vivo imaging, spleen volume, cardiac function and relaxometry were performed as described in L. Jackson et al. Animals were anaesthetized with a mixture of isoflurane and oxygen with physiological measurements used to maintain the depth of anaesthesia Cardiac function was assessed with a gradient echo cine MRI sequence with a temporal resolution of 5.

The left ventricular blood pool was segmented at systole and diastole using Segment v1. To achieve a statistically meaningful result, for each group we aimed to have at least six mice pups in each group, born by more than three different dams, in order to have more than three independent biological replicates Graph Pad Prism Version 7. Modell, B. World Health Organ.

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Rund, D. Rachmilewitz, E. How I treat thalassemia. Weatherall, D. Keynote address: The challenge of thalassemia for the developing countries.

Gene Therapy for Immunodeficiency Due to Adenosine Deaminase Deficiency | NEJM

Wilber, A. Blood , —26 Pawliuk, R. Correction of sickle cell disease in transgenic mouse models by gene therapy. Science , —71 Breda, L. PLoS One 7 , e Ye, L. Induced pluripotent stem cells offer new approach to therapy in thalassemia and sickle cell anemia and option in prenatal diagnosis in genetic diseases.