United States Patent:   6,020,144

Assignee:  Symbiontics, Inc. (Wellesley, MA); Presidents and Fellows of Harvard University (Cambridge, MA)

Filed:  October 23, 1996

Disclosed herein are novel medical devices, particular well-suited for sustained delivery of therapeutically-significant substances. Also disclosed are methods of making and using these delivery devices. Using these devices and methods the present invention teaches sustained, targeted and reversible delivery of immunostimulating agents, as well as therapeutic agents such as enzymes, hormones and neurotransmitters, to name but a few.

SUMMARY OF THE INVENTION

A novel approach to the delivery of drugs and other therapeutic substances has now been discovered. The present invention uses genetic mutations to exploit certain innate characteristics of a group of unicellular organisms known as protozoa. By genetically modifying these organisms in accordance with the present invention, the skilled practitioner can now use these organisms as improved devices for the sustained delivery of drugs and other substances.

The rationale for using these organisms as delivery devices includes the following: First, these organisms have evolved a sophisticated ability to infect a host and evade their host's immune defenses. Consequently, these organisms can persist in their host in an undetected and undisturbed state for long periods of time. As taught herein, this characteristic can be exploited to achieve sustained, in vivo delivery of a drug or other therapeutic. Moreover, by practicing the genetic manipulations disclosed herein, an organism's ability to persist in its host can be closely controlled. In fact, an organism can be engineered so that it can be subsequently eliminated on demand. Furthermore, the number of organisms in the host as well as the dose of drug or substance delivered to the host can also be closely controlled using the present invention.

Second, these organisms have evolved very specific, natural tissue preferences. Consequently, these organisms reside and persist in specific tissues in their respective hosts. Moreover, protozoa as a group reside in virtually every tissue or organ of vertebrates and invertebrates. As taught herein, this characteristic can be exploited to achieve targeted, tissue-specific delivery of a drug or other therapeutic substance. Currently, the only available type of targeted, sustained in vivo delivery relies on gene therapy, however, gene therapy cannot be similarly controlled, is often viral-vector mediated, and can result in undesirable and/or permanent alteration of the recipient's genome. Thus, the teachings of the present invention overcome the limitations of gene therapy, as well as overcome numerous problems associated with more conventional drug therapy methods, such as unnecessary systemic exposure, toxicity, poor transportability, degradation and stability to name but a few.

Accordingly, the teachings of the present invention provide a device for delivery of a drug or therapeutic substance which is sustained, targeted, reversible and virus-free, without necessarily exposing the recipient to permanent genetic alterations. In one aspect, the present invention features a sustained delivery device comprising an isolated, conditionally defective unicellular organism expressing a therapeutically-significant substance. The genome of this organism is genetically altered to lack a naturally-occurring nucleotide sequence defining a genetic locus responsible for a selectable phenotype, and encode an expression product for sustained delivery. In some embodiments, the unicellular organism is a diploid organism. In other embodiments, the organism is an asexual diploid organism. In certain currently preferred embodiments, the organism is a protozoa, more preferably a parasitic protozoa. The expression product of the present device can be encoded by a heterologous gene. Currently preferred are genes encoding a hormone, enzyme or neurotransmitter, however, any substance of therapeutic significance is contemplated. In other embodiments disclosed herein, the device can comprise an exogenous marker gene. For purposes explained herein, a currently preferred selectable phenotype is one associated with a conditional defect in metabolic function such as, but not limited to, conditional auxotrophy.

In yet another currently preferred embodiment, the above-mentioned conditionally defective organism has a selectable phenotype due to the excision of a naturally-occurring genetic locus from its genome. In this embodiment, the organism is a transfectant, the genome of which comprises transfected DNA including a nucleotide sequence free of a marker gene and which is complementary to a wild-type nucleotide sequence flanking the locus in the wild-type organism, wherein the genetic locus is excised by homologous recombination with the transfected DNA. In a most currently preferred embodiment, the transfected DNA further comprises a nucleotide sequence defining a heterologous gene. Additionally, the present invention contemplates that a selectable phenotype can be generated by other genetic alterations such as, but not limited to, alterations achieved using transposon technologies. Typically, transposons cause loss of a genetic locus by interrupting the naturally-occurring nucleotide sequence, which can generate a selectable phenotype suitable for use with the present invention.

In a second aspect, the invention features a method of providing sustained delivery of an expression product to a host comprising the step of administering any one of the above-described sustained delivery devices to the host. In a currently preferred embodiment, the method of the invention further involves the step of controlling the detectable amount of device comprising the above-described organisms or expression product produced by the organisms. A currently preferred host is a mammal, although the present invention contemplates that any metazoan organism is a suitable host, including plants, insects and mammals.

In a third aspect, the invention features a method for producing any one of the above-described sustained delivery devices. The currently preferred method comprises the steps of excising a naturally-occurring genetic locus from the genome of a unicellular organism. This can be accomplished by transfecting the organism with DNA comprising nucleotide sequences complementary to wild-type sequences flanking the locus under conditions which promote excision of the locus, and then selecting for a conditionally defective phenotype generated by loss of the genetic locus. In a preferred embodiment particularly useful for delivery of a therapeutic substance, the transfecting step contemplates transfecting with DNA further comprising a heterologous gene. As described above, this heterologous gene can encode any therapeutically-significant substance such as, but not limited to, an enzyme, hormone or neurotransmitter. In another currently preferred embodiment, the transfecting step involves transfecting with DNA further comprising an exogenous marker gene. Yet another preferred method relies on transposon technologies to induce the loss of a genetic locus and the appearance of a selectable phenotype. It is understood that that any device prepared in accordance with these methods is within the scope of the present invention.

In short, the invention provides the art with a heretofore unappreciated method of producing unique devices for sustained delivery of therapeutically-significant products to a mammal. Moreover, in accordance with present teachings, these delivery devices can be controlled to provide effective dosages of immunostimulators and/or expression products with therapeutic/pharmaceutical benefits. Furthermore, these delivery devices can be targeted to specific tissues. Finally, as will be appreciated by the skilled artisan, the devices and methods of making and using the same disclosed herein can be used in human medical and veterinary applications, as well as insect and plant applications.

Claim 1 of 17 Claims

1. A method for producing a device for administration to a host comprising the steps of:

a) transfecting a Leishmania organism with a nucleic acid comprising nucleotide sequences that correspond to naturally-occurring nucleotide sequences flanning each end of a genetic locus in the genome of said organism, under conditions to promote excision of said genetic locus from said genome;

b) selecting for a transfected Leishmania organism that lacks said genetic locus by selecting for a phenotype tat results from the excision of said genetic locus, without selecting for a phenotype encoded by a marker gene; and

c) isolating the transfected Leishmania from step b) without using a phenotype encoded by a marker gene.

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