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HALEY SCHANDELMIER
In utero gene therapy (IUGT) to the fetal hematopoietic compartment could be used to treat congenital blood disorders such as β-thalassemia. A humanised mouse model of β-thalassemia was used, in which heterozygous animals are anaemic with splenomegaly and extramedullary hematopoiesis. Intrahepatic in utero injections of a β globin-expressing lentiviral vector (GLOBE), were performed in fetuses at E13.5 of gestation. We analysed animals at 12 and 32 weeks of age, for vector copy number in bone marrow, peripheral blood liver and spleen and we performed integration site analysis. Compared to noninjected heterozygous animals IUGT normalised blood haemoglobin levels and spleen weight. Integration site analysis showed polyclonality. The left ventricular ejection fraction measured using magnetic resonance imaging (MRI) in treated heterozygous animals was similar to that of normal non-β-thalassemic mice but significantly higher than untreated heterozygous thalassemia mice suggesting that IUGT ameliorated poor cardiac function. GLOBE LV-mediated IUGT normalised the haematological and anatomical phenotype in a heterozygous humanised model of β-thalassemia.
Possible curative options that show promise are (i) the autologous correction of β-thalassaemia patient HSCs through gene augmentation studies mediated by β-globin expressing lentiviral vectors and (ii) reactivation of the endogenous HBG through lentiviral-mediated gene silencing of HBG silencer genes (e.g. BCL11A)6.
Vectors need to be assessed for safety and shown to have low oncogenic potential, in the actual biological system since the integration profile of the same vector may differ in different cell types, ex vivo and in vivo and during different developmental stages, before gene therapy application33. The safety in such system needs to be addressed34.
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 antigen47. 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 IUGT48 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 term49. Any manipulation of liver HSCs should have a long-term effect, ideally permanent, especially if these cells migrate to the thymus47. Intrahepatic fetal gene transfer to haematopoietic progenitors has also been demonstrated in primates where the maximum effect was achieved using lentiviral vectors50.
The humanised mouse model of beta thalassaemia14,15,17,19 that we used is unique in that the fetal mice produce human gamma-globin (human fetal haemoglobin) in utero15, 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 transfusions16. This model allows human beta-globin producing vectors to be tested in utero12,13,54,55, 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-natally45,56.
In this study, we show that targeting the specific stem cell niche66,67 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. found that the intravenous route was most successful for achieving rapid diffuse donor cell population of the fetal liver after in utero hematopoietic cell transplantation in mice68. 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 vector13,18. This can also be confirmed that GLOBE vector is currently used in Phase II clinical trial, using autologous stem cell/gene therapy approach in adult patients69. 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 vectors29. This also supports the use of an ex-vivo gene therapy approach using amniotic fluid stem cells or other highly efficient HSC progenitors70.
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 effect71. 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 immaturity72,73,74,75. 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 birth76 but could be better targeted in utero77.
One of the main obstacles of postnatal gene therapy is the development of an immune response against the transgenic protein or the vector78. 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 liver78,79,80,81. Delivering foreign protein to the fetus can provide an advantage of immune tolerance during fetal life, a concept first suggested more than 60 years ago82,83. 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 time84,85,86,87,88. 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. Using the BB305 vector, it reduced or eliminated the requirement for blood transfusions in 22 patients with severe β-thalassemia. No complications or reactions to the vector were reported18.
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GloVe is an unsupervised learning algorithm for obtaining vector representations for words. Training is performed on aggregated global word-word co-occurrence statistics from a corpus, and the resulting representations showcase interesting linear substructures of the word vector space.
In order to capture in a quantitative way the nuance necessary to distinguish man from woman, it is necessary for a model to associate more than a single number to the word pair. A natural and simple candidate for an enlarged set of discriminative numbers is the vector difference between the two word vectors. GloVe is designed in order that such vector differences capture as much as possible the meaning specified by the juxtaposition of two words.
The underlying concept that distinguishes man from woman, i.e. sex or gender, may be equivalently specified by various other word pairs, such as king and queen or brother and sister. To state this observation mathematically, we might expect that the vector differences man - woman, king - queen, and brother - sister might all be roughly equal. This property and other interesting patterns can be observed in the above set of visualizations.
As one might expect, ice co-occurs more frequently with solid than it does with gas, whereas steam co-occurs more frequently with gas than it does with solid. Both words co-occur with their shared property water frequently, and both co-occur with the unrelated word fashion infrequently. Only in the ratio of probabilities does noise from non-discriminative words like water and fashioncancel out, so that large values (much greater than 1) correlate well with properties specific to ice, and small values (much less than 1) correlate well with properties specific of steam. In this way, the ratio of probabilities encodes some crude form of meaning associated with the abstract concept of thermodynamic phase.
The training objective of GloVe is to learn word vectors such that their dot product equals the logarithm of the words' probability of co-occurrence. Owing to the fact that the logarithm of a ratio equals the difference of logarithms, this objective associates (the logarithm of) ratios of co-occurrence probabilities with vector differences in the word vector space. Because these ratios can encode some form of meaning, this information gets encoded as vector differences as well. For this reason, the resulting word vectors perform very well on word analogy tasks, such as those examined in the word2vec package.